methods for modifying a predetermined target nucleic acid sequence and host cell

专利摘要:
  METHODS FOR MODIFYING A NUCLEIC ACID-PREDETERMINATED TARGET SEQUENCE AND HOST CELLThe present invention provides methods for modifying a predetermined nucleic acid sequence. A programmable nucleoprotein molecular complex containing a polypeptide fraction and a specificity-promoting nucleic acid (SCNA) is provided that is assembled in vivo, in a target cell, and is capable of interacting with the predetermined target nucleic acid sequence. The programmable nucleoprotein molecular complex is able to specifically modify and / or edit a target site within the target nucleic acid sequence and / or modify the function of the target nucleic acid sequence. The present invention also relates to a host cell having genetic modification at a predetermined target site.
公开号:BR112014014105A2
申请号:R112014014105-3
申请日:2012-12-16
公开日:2020-10-27
发明作者:Yoel Moshe Shiboleth；Dan Michael Weinthal
申请人:Targetgene Biotechnologies Ltd；
IPC主号:

专利说明:

[001] [001] The present invention relates to compositions and methods of addressing and modifying nucleic acid sequences using a programmable molecular complex.
[002] [002] The main area of interest in biology and medicine is the targeted alteration of genomic nucleotide sequences. These changes include insertion, deletion and replacement of endogenous chromosomal nucleic acid sequences. Attempts have been made to genomic alteration in the past with the use of different techniques.
[003] [003] Gene addressing is a desired biotechnological tool for the manipulation of the genome or for the functional modification of the genome. Gene addressing can induce a change in a specific genomic site that may or may not be related to the coding sequences.
[004] [004] In a gene addressing event, a previously defined endogenous gene, or other previously defined endogenous nucleic acid sequence, is addressed for cleavage resulting in deletion, mutation, insertion or substitution or is addressed for chemical modification through modification of the addressed gene. An advantage of gene addressing in relation to the production of unaddressed transgenic organism is the possibility of modifying or excluding existing genomic sequences without the insertion of foreign DNA, or alternatively, positioning a foreign donor DNA, by insertion or replacement, at a predefined locus. This ability to manipulate a sequence in this way without superfluous sequences is advantageous, since these are not desired by breeders, farmers, consumers and regulatory agencies, and although many techniques
[005] [005] The strategies for gene addressing in eukaryotic beings depend on two mechanisms for repairing the breakdown of cellular dsDNA: the repair pathways of homologous recombination (HR) and non-homologous recombination (NHEJ). In NHEJ, gene insertions depend on whether there is a break in the dsDNA that can occur randomly (for example, through radiation or oxidative damage) or be addressed by a nuclease, such as the TALE nuclease (TALEN), meganuclease or nuclease zinc finger (ZFN). HR can be induced by dsDNA breaks. In HR, a break in the dsDNA is not essential, but it can increase efficiency when close to the recombination site.
[006] [006] Extensive research has been conducted on the addressing of gene mediated by HR, which works with great utility in various organisms, such as bacteria, yeast and primitive plants, moss. HR has also been used in higher organisms, such as, drosophila, mice, and humans. The rates of HR in these organisms are around 10º-6, and can be increased beyond 10º-2, in assisted HR, creating a specific DSB of the gene. Low rates of transformants are one reason why these methods are not prevalent in gene therapy or breeding programs.
[007] [007] Various techniques for modifying nucleic acids in vivo have been suggested and can be divided into methods based on enzymes or nucleotides. In general, enzyme-based methods use a DNA binding protein that has the desired catalytic activity and the ability to bind to the desired target sequence through a nucleic acid-protein interaction similar to enzymes restriction. Examples include meganucleases, which are synthetic or natural rare-sequence cutting enzymes, zinc finger nucleases (ZFNs) or transcription activator-like nucleases (TALENs) that contain the subunit.
[008] [008] For nucleotide-based methods, nucleic acids are supplied to the organism and endogenous processes cause DNA repair or gene addressing through unattended homologous recombination or integration of the oligonucleotide into the genome. These nucleic acids can be supplied using viral vectors, plasmid vectors, T-DNA vectors and double-stranded DNA oligonucleotides. Shorter nucleotides called triple helix-forming oligonucleotides (TFOs) are used for oligonucleotide-based mismatch repair, and can perform the repair of point mutations or repair of up to 4 nucleotides. There is ample evidence that these methods also depend on the formation of DSBs, which can be random, randomly induced or locally induced through enzymatic or chemical modification with enzymes or reactive chemicals covalently linked to the supplied nucleic acid. Double strand breaks (DSB) in DNA are required for HR. Existing specific DSBs are not essential, although they increase efficiency. Natural DNA breaks are randomly located and rare, so efficiency must be low (107-6). DSBs can be randomly induced with ionizing radiation or oxidizing chemical substances, increasing efficiency in preventing genotoxicity. In the improvement of this system, the repair or assisted HR were performed in the past with the use of non-enzymatic DNA cleavage assisted by the chemical modification of the nucleic acid terminal. These modifications include photoactivable EDTA-Fe or Psoralen and were used to produce a sequence specific DSB in dsDNA when incorporated in vitro to form a triple helix. An additional method uses oligonucleotides, or modified oligonucleotides, derived from single-stranded DNA (ssDNA), also known as “synthetic single-stranded small oligodexoxynucleotides (ODNs or SSODNs). However, although oligonucleotide-based methods can result in relatively efficient point mutations in mammalian cell genomes, they are restricted to this mode of editing.
[009] [009] The oligonucleotide-enzyme conjugates are a combination of two methods comprising a nucleic acid covalently linked in vitro to a catalytic enzyme before delivering the conjugate to the body. These methods, in
[010] [010] Unconjugated oligonucleotide-protein systems have also been used to cleave an ssDNA substrate. In this system, a Class-llS Restriction Enzyme, Fokl, which cleaves outside its recognition site, was used in vitro, together with a clamp-forming oligonucleotide that reconstitutes the Fokl recognition sequence, with a Pollk enzyme and dNTPs for create a double-stranded section of DNA initiated by the oligonucleotide to be cleaved. In this system, not only is the intended sequence cleaved, but any naturally occurring FokI site will be recognized and the sequence adjacent to it will be cleaved. As Fokl has only one recognition site with 5 nucleotides, this implies that there are thousands of potential cleavage sites in an entire genome, making this system useless for editing the genome.
[011] [011] In higher plants and humans, in contrast to other organisms where HR can be used for gene addressing, the NHEJ pathway is the predominant endogenous mechanism. The vegetable DNA repair machine does not allow HR between donor and chromosome DNA. In fact, it is widely accepted that foreign donor DNA molecules, which are generally delivered through Agrobacterium-mediated genetic transformation, are recognized via non-homologous recombination (NHEJ), which causes random integration throughout the genome host. The most common plant transformation methods, therefore, are not considered gene addressing, because in these methods, the sequences are inserted randomly into the genome, and as an unwanted side effect, the existing gene can collapse, and in general they are inserted in multiple copies, or contain unwanted remnants of the bacterial sequence, plasmid or marker.
[012] [012] Methods for inducing specific dsDNA breaks, useful for assisted HR and directed NHEJ, use the expression of nucleases in vivo. These include rare-sequence cut-off nucleases, such as chimeric meganucleases or meganucleases, derived from homing endonucleases, custom recombinant Zinc Fingers (ZFNs), or recombinantly produced TAL effector nucleases customized. In these methods, recognition of the cleaved target site is achieved by the interaction of a subunit or protein domain that naturally recognizes a specific nucleotide sequence, or is developed specifically to recognize a specific nucleotide sequence and is not based on in base pairing or polynucleotide-polynucleotide hybridization. For example, zinc finger nucleases are chimeric proteins, constructed as hybrids between the Fokl nuclease subunit and synthetic zinc finger (ZF) domains. Zinc finger nucleases do not contain a nucleic acid component. ZFNs are designed to specifically recognize nucleotide triplets by combining several ZF motifs. ZFNs cannot be constructed to recognize all sequences because of their inherent ability to recognize only a limited subset of nucleotide triplets. The use of ZFN heterodimers, therefore, two different ZFNs, which are inactive as a monomer, are delivered concurrently, have a positive effect on specificity, although this makes the design even more complex and reduces the choice of target sequences. ZFNs were also used to create artificial transcription factors to activate and repress genes, to alter gene regulation. However, these zinc finger-based transcription factors cannot bind to all sequences, being limited in terms of recognition site length and restricted to several specific trinucleotide motifs, so they cannot be used to activate or suppress possible genes.
[013] [013] For example, Schierling et. al., reveal an unusual platform of zinc finger nucleus with a sequence-specific cleavage module. For example, Eisenschmidt K, et. al. They reveal a restriction endonuclease programmed for highly specific DNA cleavage. For example, WO 2006/027099 deals with enzyme conjugates with programmable specificity, which react in a highly specific way with DNA.
[014] [014] Kubo et. al., for example, reveal the control of intracellular oligonucleotide delivery by signal peptides and gene expression in human cells. Jinek et. al., reveal a programmable Double DNA Oriented DNA endonuclease in adaptive bacterial immunity.
[015] [015] WO 2012/129373, for example, addresses methods for producing a locus of complex transgenic characteristics.
[016] [016] However, the need remains unmet in the art for safe, reliable, modular and economical compositions and methods that allow the modification and specific addressing of target nucleic acid sequences in vivo.
[017] [017] The present invention provides compositions and methods for addressing and modifying nucleic acid sequences, in vivo or in vitro. According to some modalities, the unusual composite programmable molecular complex (nucleoprotein complex) provided here is used to edit or functionally adapt a predetermined target nucleic acid sequence precisely,
[018] [018] In some embodiments, the molecular complex disclosed here is used for gene addressing and / or functional modification of the addressed gene that includes, but is not limited to, generation of breaks in one or two strands of the target nucleic acid to initiate gene mutation, deletion, gene replacement, and integration of a foreign nucleic acid molecule, or for its functional chemical, conformational or biological modification.
[019] [019] According to some modalities, the molecular complex disclosed herein comprises a) a chimeric polypeptide (which can be encoded by a polynucleotide molecule), the chimeric polypeptide comprising: (i) a functional (effector) domain (FD) able to modify a target site; and (ii) a binding domain (LD); and (b) a specificity-enhancing nucleic acid (SCNA), the SCNA comprising: (i) a nucleotide sequence complementary to a region of a target nucleic acid flanking the target site; and (ii) a recognition region capable of specifically attaching to the polypeptide binding domain; and thus the assembly of the polypeptide and the SCNA within a host / target cell forms a programmable and functional molecular nucleoprotein complex capable of specifically modifying the target nucleic acid at the target site.
[020] [020] In some embodiments, the present invention provides an advantageous composition comprising a protein effector module (or a nucleic acid molecule encoding it) and a programming / addressing nucleic acid module that can be assembled autonomously in live forming a molecular nucleoprotein complex that modifies the specific active nucleic acid. In this complex, the nucleic acid, here also called “programming fraction”, ““ programming oligonucleotide ”or“ specificity-promoting nucleic acid ”(SCNA) provides the specificity and binding capabilities of the molecular complex to the target nucleic acid through the base pairing of said specificity-providing nucleic acid and a target nucleic acid. The protein module or effector component of this complex is designed to bind / unite / attach to specificity-promoting nucleic acid through a chemical fraction attached to the oligonucleotide, a modification of a nucleotide or nucleotides in the oligonucleotide, a specific recognition sequence in the oligonucleotide, and the like, or combinations of those cited. From the vantage point, the compositions and methods disclosed here confer higher specificity with a wide range of desired target sequences, are less genotoxic, modular in assembly, reliable, use a single platform without customization, are practical to use independent from specialized essential facilities, and have a shorter development time span and reduced costs.
[021] [021] The activity of the protein module can result in the modification of the target nucleic acid sequence and / or in the functional modification of the target nucleic acid. The target nucleic acid modification may include, among other things: mutation, deletion, insertion, substitution, binding, digestion, nicking, methylation, acetylation, binding, recombination, helical exhalation, chemical modification, labeling, active - tion, and inactivation or any of its combinations. Functional modification of the target nucleic acid can induce, among other things: changes in transcription activation, transcription inactivation, alternative splicing, chromatin rearrangement, pathogen inactivation, virus inactivation, alteration of the cellular localization, acid compartmentalization nucleic, and the like, or combinations thereof. Any editing action or other modification carried out by the protein fraction is directed or directed to a specific (predefined) target nucleic acid through its binding to the specificity-providing nucleic acid. From an advantageous point of view, the use of each unique type of protein can be associated with an unlimited assortment of nucleotide sequences of nucleic acids that provide specificity concomitantly or separately, to allow similar action in different sections of the acid target nucleic acid. This makes it possible to overcome the disadvantages of prior art methods by providing reliable and economical versatile methods and compositions for modifying the targets of the predetermined nucleic acid sequence. Thus, if used in a depository or organism, only one type of protein should be provided together with any combination or multiplicity of nucleic acid types that provide specificity. This also includes the possibility of the concomitant use of more than one type of protein component with more than one type of nucleic acids that provide specificity.
[022] [022] According to some modalities, the complex disclosed here is modular and can be assembled autonomously inside a target cell, either in vivo or in vitro, allowing the supply of a type of protein fraction at once and with one or multiple oligonucleotides providing concomitant specificity. In addition, in some embodiments, the protein component can be delivered to a desired cell (s) and expressed in vivo, awaiting delivery of any appropriate SCNA at a future time. In some embodiments, the protein component and the SCNA can be delivered simultaneously, or essentially simultaneously. In this way, the combination of the protein component and the SCNA, preferably within the desired target cell, can induce breaks in the specific genomic double strand (DSBs), or any other modification of nucleic acid desired, in vivo. The methods of the present invention are not restricted to the introduction of point mutations in the target nucleic acid, since the molecular complex can address any nucleic acid sequence or pair of sequences, cut / restrict / cleave areas very close to them, and consequently exclude a large or small section of nucleic acid, or cut / restrict / cleave the sequence in order to initiate a removal, or an insertion, or a replacement of any nucleic acid sequence.
[023] [023] Under the advantageous aspect, the present invention reveals for the first time in its modalities the expression of a component of the protein in vivo and its binding / attachment to the SCNA (s) through self-assembly in vivo to form a molecular complex in vivo, without the need for prior covalent / chemical bonding between the protein fraction and the targeting nucleic acid.
[024] [024] According to some modalities, as an unscheduled protein component (that is, a protein not attached / linked to a programming oligonucleotide) has no affinity, or this affinity is very low, target nucleic acids, makes the possibility of achieving greater specificity and safety and less genotoxicity is advantageous. As detailed above, the effector or catalytic domain of the protein component is only active after dimerization, and therefore at least two programming oligonucleotides (SCNAs) must bind to the target flanking sequences to cause dimerization and activation of the protein. Two sufficiently long programming oligonucleotides can provide the extremely high theoretical specificity required in highly complex genomes by creating extensive complementarity with the binding sites. Since the expressed, unscheduled protein has no affinity for the target nucleic acid, it does not bind, and / or modify the target nucleic acid. Thus, in applications where, for example, programming oligonucleotides are delivered / supplied separately to the target cell (which already expresses the unscheduled protein component), or in conditions where oligonucleotides are excluded from the cell Target (for example, through dilution or degradation), nonspecific cleavage cannot occur, which increases safety and reduces genotoxicity.
[025] [025] Thus, according to the modalities of the present invention, both addressed non-homologous recombination (NHEJ) and assisted homologous (HR) recombination can be used specifically and programmatically to obtain one or more of the following events:
[026] [026] Mutate a DNA sequence cleaving its interior, creating a double strand break (DSB), which will be degraded in part by endogenous nucleases and rewired by the endogenous NHEJ DNA repair mechanism to create a deletion within the frame and / or a DNA frame shift mutation. Contrary to T-DNA or transposon insertion lines in plants, this method of deleting or mutating an endogenous gene does not allow any foreign DNA to remain and the plant can be called non-transgenic according to some definitions. In NHEJ, one or more nucleotides can also be added to the DSB in an endogenous mechanism not yet characterized, obtaining essentially the same effect of displacement of the reading frame or mutation.
[027] [027] Delete an extension of the DNA sequence by cleaving two flanking sequences, which will be rewired by the endogenous NHEJ DNA repair mechanism, or by assisted HR cleaving at or close to the sequence to be deleted and providing a donor DNA which is subsequently recombined in the target, and which contains sequences flanking the sequence to be excluded in the target.
[028] [028] Inserting a donor nucleic acid into a DSB by cleaving a target nucleic acid and providing a Donor DNA that will be linked directly to the gap by the NHEJ mechanism, or preferably, providing a donor that has homology to the ends of the gap to be recombined and linked in the gap by assisted HR.
[029] [029] Replace a target nucleic acid sequence by cleaving both flanking sequences, and providing a donor nucleic acid to be inserted, which will be linked within the target flanking sequence, either by NHEJ or, preferably,
[030] [030] According to some modalities, and without being bound by theory or mechanisms, the advantages of the compositions and methods disclosed here, include the creation of a scheme for the construction of a general enzyme complex that can address a selection unlimited strings. Once a protein component has been optimized for a specific purpose (for example, dsD-NA cleavage), this same protein can be used with an unlimited selection of programming nucleic acid (SCNA) sequences. Thus, the diversity of the target sequences that will be affected is obtained by the design of the SCNA, without the complex and demanding need for a new design and optimization of the protein, inherent to other methods known in the art, such as TALENs, ZFNs and Meganucleases, where the protein itself must be altered and adapted to each target sequence. The design and preparation of synthetic SCNAs is relatively simple, fast and economical. It is also possible, in some embodiments of this invention, to produce SCNAs in vivo, escaping the need to deliver chemically synthesized SCNAs to a cell. In addition, SCNAs can be designed to pair bases with virtually any desired target sequence, and thus address the molecular complex to virtually any target sequence. In addition, several target sequences can be used in the same cell concurrently. For example, in editing functions that require more than one cleavage site, for example, deletion or replacement of specific extensions of nucleic acid, simply providing four different SCNAs and a fraction of protein.
[031] [031] According to some modalities, a nucleoprotein composition is thus provided to modify a predetermined target site in a target nucleic acid sequence in a target cell, the composition comprising: a polynucleotide molecule that encodes a polypeptide, or a polypeptide,
[032] [032] In some modalities, the functional domain comprises a catalytic domain. In some embodiments, the polypeptide further comprises a subcellular localization domain.
[033] [033] In some embodiments, the modification of the target nucleic acid is selected from: mutation, deletion, insertion, substitution, ligation, digestion, promoting double strand breaking, nicking, methylation, acetylation, ligation, recombination, propeller exhalation, chemical modification, marking, activation and inactivation.
[034] [034] According to some modalities, SCNA comprises a nucleic acid molecule selected from the group formed of a single-stranded DNA, a single-stranded RNA, a double-stranded RNA, a modified DNA , a modified RNA, a blocked nucleic acid (LNA) and a peptide-nucleic acid (PNA) or combinations thereof.
[035] [035] In some modalities, the SCNA recognition region comprises
[036] [036] In some modalities, the attachment / link / association between the modification in the SCNA and the link domain results from an interaction of a selected link pair between non-covalent interaction of a selected link pair between, but not limiting to: Biotin-Avidin; Biotin-Streptavidin; biotin-modified forms of avidin; protein-protein; protein-nucleic acid interactions; linker-receptor interactions; binder-substrate interactions; antigen-antibody; antigen-single chain antibody; antibody or single chain-hapten antibody; hormone-hormone binding protein; receptor-agonist; receptor-receptor antagonist; IgG protein A; enzyme-enzyme cofactor; enzyme-enzyme inhibitor; Single-stranded DNA-VirE2; StickyC - dsDNA; RISC - RNA; nucleic acid of the viral coat protein; anti-fluorescein single-chain variable fragment antibody (anti-FAM ScFV) - Fluorescein; anti-DIG single-chain variable fragment immunoglobulin (scFv) (DIG-ScFv) - Digoxigenin (DIG) and Agrobacterium VirD2-VirD2 binding protein; and any variants thereof.
[037] [037] In some embodiments, the SCNA recognition region comprises a nucleotide motif capable of specifically attaching / binding / associating to the chimeric protein binding domain. In some embodiments, the attachment / association / attachment between the nucleotide motif and the binding domain is selected from, but not limited to: Finger zinc protein-finger zinc motif; domain of restriction enzyme recognition-restriction enzyme recognition sequence; DNA binding domain of the DNA motif-transcription factor; repressor-operator; Leucine zipper — promoter; helix-loop-helix-E box domain; RNA binding motifs comprising domains of the argin rich motif, ab protein domains, RNA recognition motif domains (RRM), K-Homology domains, RNA binding motifs with Double Filament, Zinc Fingers binding to RNA, and RNA Addressing enzymes - cognate specific RNA sequence; HIV-rev- Stem IIB protein of the HIV rev response element (RRE); Tat main domain of bovine immunodeficiency virus (BIV) of link-loop 1 of the trans-action response element (TAR) sequence
[038] [038] According to some modalities, a method is provided for modifying a predetermined target site within a target nucleic acid sequence using a programmable nucleoprotein molecular complex, the method comprising the steps of: a) delivering a nucleic acid sequence that encodes a programmable chimeric protein (polypeptide) or protein (polypeptide) to a host cell; b) delivering a specificity-promoting nucleic acid molecule (SCNA), or a nucleic acid encoding SCNA to said host cell; c) linking said chimeric protein to SCNA, directing the chimeric protein to the predetermined target nucleic acid sequence within the host cell, to form an active programmed nucleoprotein complex; and d) enabling the modification of the predetermined target site of the target nucleic acid sequence by said active programmed nucleoprotein molecular complex.
[039] [039] In some embodiments, a method is provided for modifying a predetermined target site within a target nucleic acid sequence using a programmable nucleoprotein molecular complex, the method comprising the steps of: . delivering a nucleic acid sequence encoding a programmable chimeric polypeptide to a host cell, said chimeric polypeptide comprising: i. a functional domain capable of modifying said target site, the functional domain being devoid of a specific nucleic acid binding site; and ii. a binding domain that is capable of interacting with a specificity-promoting nucleic acid, wherein the binding domain is devoid of a specific target nucleic acid binding site; B. deliver a specificity-enhancing nucleic acid molecule
[040] [040] in which the expression of the polypeptide in the cell that hosts the SCNA makes it possible to attach said chimeric polypeptide to the SCNA, forming a programmed active nucleotide complex, thereby directing the chimeric polypeptide to the predetermined target nucleic acid sequence in inside the host cell, allowing the modification of the predetermined target site of the target nucleic acid sequence by said active programmed nucleoprotein molecular complex.
[041] [041] In some embodiments, the target nucleic acid is DNA. In some modes, the target DNA is genomic DNA. In some embodiments, the target nucleic acid sequence is an extrachromosomal nucleic acid sequence. In some embodiments, the target extrachromosomal nucleic acid sequence resides in an organelle selected from the group formed by mitochondria, chloroplasts, amyloplasts and chromoplasts. In some embodiments, the target nucleic acid sequence is a viral nucleic acid sequence. In some embodiments, the target nucleic acid sequence is a prokaryotic nucleic acid sequence. In some embodiments, the target nucleic acid sequence is a synthetic nucleic acid sequence.
[042] [042] In some modalities, the modification is selected between mutation, deletion, insertion, substitution, bonding, digestion, promoting double filament breaking, nicking, methylation, acetylation, bonding, recombination, despiration of propeller, chemical modification, marking, activation and inactivation.
[043] [043] In some embodiments, the chimeric protein (polypeptide) comprises a fraction of protein that has nucleic acid-modifying activity. In some embodiments, the chimeric protein comprises a protein fraction that has a functional nucleic acid modifier, in which the functional modification is selected from the group formed by transcription activation, transcription inactivation, silencing of the RNA transcript, alternative splicing of RNA, chromatin rearrangement, cell parasite and virus inactivation and alteration of the cell location or compartmentalization of the said target nucleic acid sequence.
[044] [044] In some embodiments, the SCNA comprises a molecule selected from the group consisting of a single-stranded DNA, a single-stranded RNA, a double-stranded RNA, a modified DNA, a modified RNA, a blocked nucleic acid (LNA) and a peptide-nucleic acid (PNA) or combinations thereof. In some embodiments, the SCNA comprises a specificity-enabling sequence configured specifically to interact with the target nucleic acid. The interaction between the SCNA and the target nucleic acid occurs through base pairing, selected from the group formed by an integral double helix base pairing, a partial double helix base pairing, an integral pairing of triple helix base, a partial pairing of triple helix base, and D loops or branched shapes, formed by said base pairing.
[045] [045] In additional modalities, the SCNA comprises a recognition region configured to associate / link / attach to a binding domain of the chimeric protein. In some embodiments, the recognition region comprises a modification selected from the group formed by modification of the 5'-terminal, modification of the 3'-terminal, and internal modification. The modification can be selected from, but not limited to, modification of the nucleotide, Biotin, Fluorescein, Amine linkers, oligopeptides, Aminoalyl, a dye molecule, fluorophores, Digoxigenin, Acridite, Adenylation, Azide, NHS-ester, Colesteril-TEG, Alcinos, Bioti-
[046] [046] In some modalities, the association / link / annexation between the modification in the SCNA and the link domain results from a non-covalent interaction of a selected link pair between: Biotin-Avidin; Biotin-Streptavidin; biotin-modified forms of avidin; Protein-protein interactions; protein-nucleic acid interactions; ligand-receptor interactions; binder-substrate interactions; antigen-antibody interactions; single chain antigen-antibody; antibody or single chain antibody-hapten interactions; hormone-hormone-binding protein; receptor-agonist; receptor-receptor antagonist; anti-fluorescein single chain variable fragment antibody (anti-FAM ScFV) - Fluorescein; anti-DIG single chain variable fragment immunoglobulin (scFv) (DIG-ScFv) - Digoxigenin (DIG); protein A-IgG; enzyme-enzyme cofactor; enzyme-enzyme inhibitor; Single-stranded DNA-VirE2; StickyC - dsDNA; RISC - RNA; nucleic acid of the viral coat protein and VirD2-VirD2 binding protein from Agrobacterium; and any variants thereof.
[047] [047] In some embodiments, the link / association between the nucleic acid sequence conferring specificity and the protein fraction binding domain is created covalently in vivo. In some embodiments, the covalent association of the binding domain and the SCNA results from a biological interaction of VirD2 from
[048] [048] In some embodiments, the recognition region comprises a nucleotide motif capable of interacting / attaching / binding to the binding domain of the chimeric protein. In some modalities, the interaction pair is selected from among: Zinc finger protein-zinc finger motif; domain of restriction enzyme recognition - restriction enzyme recognition sequence; DNA binding domain of the transcription factor-DNA motif; repressor-operator; Leucine-promoting zipper; domain E box-Helix-loop-helix; RNA binding motifs comprising arginine-rich motif domains, aB protein domains, RNA recognition motif (RRM) domains, K-Homology domains, Double Filament RNA binding motifs, Zinc-binding fingers RNA, and RNA Addressing Enzymes - cognate specific RNA sequence; HIV-rev- Stem IIB protein of the HIV rev response element (RRE); Tat primary domain of loop-binding bovine immunodeficiency virus (BIV) 1 of the BIV trans-action response element (TAR) sequence; Lambda phage, phi21, and Nproteins P22 - the clamps of the boxB loop at the N-use (nut) sites in their respective RNAs.
[049] [049] According to some modalities, the predetermined target nucleic acid sequence is involved in a genetic characteristic, and the modification results in changes in the transcription or translation of a genetic element, by means of a technical procedure selected from the group formed by substitution, permanent knock-out or intensification, shut-off, knock-down, and displacement of the temporary or permanent reading frame. In some modalities, the genetic characteristic is modified by editing the sequence of the genetic element itself, its regulatory sequences, regulatory genes of the gene of interest or its regulatory sequences in a regulatory chain of events.
[050] [050] According to other modalities, a nucleoprotein complex is provided, in which a physical association between the protein fraction and the nucleic acid fraction that provides specificity forms a programmed functional complex. In some modalities, the physical association between the protein fraction binding domain and the SCNA is based on a selected affinity interaction between the ligand-receptor, ligand-substrate group, hydrogen bonds, van der bonds Waals, ionic bonds and hydrophobic interaction.
[051] [051] According to some modalities, a host cell with predetermined genetic modification is provided at a predetermined target site, created by the method disclosed here. In some embodiments, the host cell can be any type of cell, such as, but not limited to: vertebrate cell, mammalian cell, human cell, animal cell, plant cell, invertebrate cell, nematode cell, insect cell and stem cell.
[052] [052] According to some modalities, a transgenic organism or knock out organism is provided, with predetermined genetic modification formed by the method described here. In some embodiments, the organism is a plant or animal.
[053] [053] According to some modalities, a method of treating a genetic disease in an organism is provided, the method comprising introducing into the cell of the organism the programmable molecular complex of nucleoprotein.
[054] [054] According to some modalities, a host cell is provided comprising: a) a polypeptide comprising: (i) a functional domain capable of modifying a target site in a target nucleic acid sequence in the cell, the functional domain being devoid of a specific nucleic acid binding site; and (il) a binding domain that is capable of interacting with a specificity-providing nucleic acid and being devoid of a specific target nucleic acid binding site; and; (b) a specificity-enhancing nucleic acid (SCNA) comprising: i. a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site; and (ii) a recognition region capable of specifically attaching to the polypeptide binding domain;
[055] [055] And so the assembly of the polypeptide and the SCNA inside the host cell forms a functional nucleoprotein complex capable of specifically modifying the target nucleic acid at the target site.
[056] [056] In some embodiments, a host cell is provided that houses: (a) a polynucleotide molecule encoding a polypeptide, the polypeptide comprising: (i) a functional domain capable of modifying a target site in a nucleic acid sequence target in the cell, the functional domain being devoid of a specific nucleic acid binding site; and (ii) a binding domain that is capable of interacting with a specificity-enhancing nucleic acid and being devoid of a specific target nucleic acid binding site; and (b) a specificity-promoting nucleic acid (SCNA) comprising: (i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site; and (ii) a recognition region capable of specifically attaching to the polypeptide binding domain; and thereby the assembly of the polypeptide and SCNA within the host cell forms a functional nucleoprotein complex capable of specifically modifying the target nucleic acid at the target site.
[057] [057] Figures 1A-B are schematic boards showing elements / components of a programmable molecular complex, according to some modalities;
[058] [058] Figures 2A-B are schematic boards showing the assembly of the programmable molecular complex, according to some modalities;
[059] [059] Figure 3 shows an example in a three-dimensional model of a molecular complex designed for the cleavage of a predefined nuclear dsDNA target sequence, according to some modalities;
[060] [060] Figures 4A-B are schematic drawings (out of scale) of the exemplary way of assembling the components of the programmable molecular complex in a target nucleic acid, according to some modalities.
[061] [061] Figure 5 is a schematic drawing showing the delivery of the programmable molecular complex to a cell using SCNAs produced in vitro, according to some modalities;
[062] [062] Figure 6 is a general diagram showing the delivery of the programmable molecular complex to a cell using an SCNA produced in vivo, according to some modalities;
[063] [063] Figures 7A-B are schematics showing non-limiting examples of the delivery of the fraction of the nucleic acid programming the molecular complex to a cell using a SCNA with single-stranded DNA produced in Agrobacterium (Figure 7A) and bacterial secretion system (Figure 7B), according to some modalities;
[064] [064] Figures 8A-B are a schematic illustration showing the delivery of the programming fraction of the programmable molecular complex to a cell using RNA SCNAs produced in Agrobacterium (Figure 8A) or by an autonomous replication vector, such as a virus (Figure 8B), according to some modalities;
[065] [065] Figure 9 shows a schematic illustration (out of scale) of a non-limiting example of a vector or delivery vehicle for the concomitant delivery of the composition comprising the components necessary for the assembly of a programmable molecular complex to a cell. eukaryotic target susceptible in a single delivery event, according to some modalities;
[066] [066] Figure 10 is a schematic illustration (out of scale) demonstrating the use of a programmed molecular complex to create a mutation in a Target nucleic acid, according to some modalities.
[067] [067] Figure 11 is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to insert one or multiple nucleotides into a target nucleic acid using a donor nucleic acid provided, according to some modalities.
[068] [068] Figure 12 is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to replace one or multiple nucleotides in a target nucleic acid using a donor nucleic acid provided, according to some modalities.
[069] [069] Figure 13 is a schematic illustration (out of scale) demonstrating the use of a programmed molecular complex to create a deletion of one or multiple consecutive nucleotides from a target nucleic acid, according to some modalities.
[070] [070] Figure 14 is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to replace one or multiple nucleotides in a target nucleic acid using a donor nucleic acid provided, according to some modalities.
[071] [071] Figure 15 shows a schematic illustration of a non-limiting example of a vector or delivery vehicle (out of scale) for concomitant delivery of the programmable molecular complex protein (PMCP) to a susceptible eukaryotic target cell together with a target sequence to test your activity, according to some modalities, and as detailed in Example 10.
[072] [072] Figure 16 shows a schematic drawing (out of scale) of parameters that empirically determine the ideal distance between the pairs of SCNA and to test the ability of different types of molecular complexes programmed to specifically cleave a target DNA, as detailed in Example 12. Detailed Description of the Invention
[073] [073] According to some modalities, compositions and methods for modifying a predetermined target nucleic acid are provided. Specifically, methods for modifying a target sequence in vivo using a composition comprising a programmable molecular complex are disclosed. The programmable molecular complex (here also called "nucleoprotein complex") comprises a fraction of protein, (here also called "programmable fraction"), and a fraction of nucleic acid, (here also called "specificity-promoting nucleic acid" ”(SCNA) or“ the programming nucleic acid ”). According to some modalities, the components of the molecular complex are assembled automatically in vivo in a living cell, organism, tissue, callus, organ or part thereof, differentiated or not, in the presence of a nucleic acid sequence (s) - target to form an active programmed functional molecular complex.
[074] [074] It is understood that the terminology used here is intended to describe particular modalities only, and is not intended to be limiting. It should be noted that, as used in the specification and in the appended claims, the singular form "one", "one", "o" and "a" includes plural equivalents, unless the context clearly dictates otherwise.
[075] [075] For the citation of numerical ranges, each intermediate number with the same degree of precision is explicitly considered. For example, for the range 6-9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6 , 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[076] [076] about
[077] [077] As used herein, the term “about” refers to +/- 10% ,.
[078] [078] administer
[079] [079] “Administer” refers to the provision of a composition or pharmaceutical agent to an individual, and includes, among other things, administration by a medical professional and self-administration.
[080] [080] "Parenteral administration" means administration not through the intestines. Parenteral administration includes, among other things, subcutaneous administration, intravenous administration, or intramuscular administration.
[081] [081] "Subcutaneous administration" means administration just under the skin.
[082] [082] "Intravenous administration" means administration into the vein.
[083] [083] "Intratumoral administration" means administration within a tumor.
[084] [084] “Chemoembolization” means a procedure in which the blood supply to the tumor is blocked surgically or mechanically and chemotherapeutic agents are administered directly to the tumor.
[085] [085] The term "antisense", as used herein, refers to nucleotide sequences that are complementary to a specific DNA or RNA sequence. The term "antisense filament" is used in reference to a nucleic acid filament that is complementary to the "sense" filament. Anti-sense molecules can be produced using any method, including synthesis by binding the gene (s) of interest in a reverse direction to a viral promoter that allows for the synthesis of a complementary filament Once inserted into a cell, this transcribed filament combines with the natural sequences produced by the cell to form duplexes. These duplexes then block both transcription and later translation. Thus, mutant phenotypes can be generated. .
[086] [086] "Autonomously replicating vectors" are defined here to comprise any natural or synthetic nucleic acid sequence capable of replicating within a host, comprising, but not limited to, viruses, modified viruses, some recombinant vectors and plasmids, replicates and intracellular parasites. cell
[087] [087] “Cell” is defined here to include any cell type, prokaryotic or eukaryotic cell, isolated or not, cultured or not, differentiated or not, and comprising a high level of cellular organization, for example, tissues, organs , calluses, organisms or parts of those mentioned. Exemplary cells include, but are not limited to: vertebrate cells, mammalian cells, human cells, plant cells, animal cells, invertebrate cells, nematode cells, insect cells, stem cells, and the like. complement
[088] [088] "Complement" or "complementary", as used herein, means the base stacking of Watson-Crick (for example, A-T / U and C-G) or Hoogsteen between nucleotides or nucleotide analogs of nucleic acid molecules. A complete or totally complementary complement can mean 100% complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. Partial complementary can mean less than 100% complementarity, for example, 80% complementarity. delivery vector
[089] [089] "delivery vector" or "delivery vectors" is any delivery vector that can be used in the present invention to bring the cell into contact or deliver the chemical agents / substances to the cells or subcellular compartments molecules (proteins or nucleic acids) needed in the present invention. This includes, among other things, transduction vectors, liposomal delivery vectors, plasmid delivery vectors, viral delivery vectors, bacterial delivery vectors, delivery and drugs vectors, chemical carriers, carrier-
[090] [090] "Dose", as used herein, means a specified amount of a pharmaceutical agent provided in a single administration. In some modalities, a dose can be administered in two or more cakes, tablets, or injections. For example, in some modalities, when subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In these modalities, two or more injections can be used to obtain the desired dose. In some modalities, a dose can be administered in two or more injections to minimize the reaction at the injection site in an individual. dosing unit
[091] [091] "Dosage unit", as used herein means, a method of supplying a pharmaceutical agent. In some embodiments, a dosage unit is a vial containing lyophilized oligonucleotide. In some modalities, a dosage unit is a bottle containing reconstituted oligonucleotide. donor nucleic acid
[092] [092] “Donor nucleic acid” is defined here as any nucleic acid supplied to an organism or receptacle to be inserted or recombined, in whole or in part, in the target sequence by DNA repair mechanisms, homologous recombination (HR), or non-homologous recombination (NHEJ).
[093] [093] "Duration", as used herein, means the length of time an activity or event lasts. In some embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent or pharmaceutical composition are administered. expression vector
[094] [094] "Expression vector", as used herein, means any nucleic acid designed to artificially encode an exogenous protein or proteins in a host cell. Examples of expression vectors include plasmid DNA, T-DNA, viral RNA, ssDNA or dsDNA, Replicons, autonomous replication vectors, linear ssDNA, linear dsDNA, phi polymerase products, RNA transcript, circular RNA, and in some applications of this invention, genomic and organellar DNA transferred to the host cell. fragment
[095] [095] "Fragment" is used here to indicate a full-length part of a nucleic acid or polypeptide. Thus, the fragment itself is also a nucleic acid or polypeptide, respectively. gene
[096] [096] "Gene", as used herein, can be a natural (for example, genomic) or synthetic gene comprising transcription and / or translation regulatory sequences and / or a coding region and / or untranslated sequences ( for example, introns, 5'- and 3 'untranslated sequences). The coding region of a gene can be a nucleotide sequence that encodes an amino acid sequence or a functional RNA, such as, tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA. A gene can also be an mRNA or cDNA corresponding to the coding regions (for example, exons and miRNA) optionally comprising 5'- and 3 'untranslated sequences linked to it. A gene can also be an amplified nucleic acid molecule produced in vitro comprising all or part of the coding region and / or 5 "- and 3 'untranslated sequences linked to the gene.
[097] [097] “Gene addressing” is used here as any genetic technique that induces a permanent change in a target nucleic acid sequence, including deletion, insertion, mutation and substitution of nucleotides in a target sequence . genomic modification
[098] [098] “Genomic modification” is used here as any modification generated in a genome or a chromosome or extrachromosomal DNA or organellar DNA of an organism as a result of gene addressing or functional modification of the gene. host cell
[099] [099] "Host cell", as used herein, can be a naturally occurring cell or a transformed cell that can contain a vector. Host cells can be cultured cells, explants, cells in vivo, and the like. Host cells can be prokaryotic cells, such as, E. Coli, or eukaryotic cells, such as, plant, yeast, insect, amphibian, or mammalian cells, such as, CHO and HeLa.
[0100] [0100] According to some modalities, said host cell is an entire cell or not, differentiated or not, in the organism, organ, tissue or callus. identity
[0101] [0101] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences display a specified percentage of the same residues over a specified region. The percentage can be calculated by ideally aligning the two sequences.
[0102] [0102] "Inhibiting", as used herein, may mean avoiding, suppressing, suppressing, reducing or eliminating. in vitro
[0103] [0103] "In vitro" is defined here as an artificial environment outside the membranes of a living organism, organ, tissue, callus or whole cell or not, differentiated or not. In some embodiments, the term in vitro does not include a viable cell. in vivo
[0104] [0104] “Ih vivo” is defined here as inside a complete or partial organism, organ, tissue, callus or cell, differentiated or undifferentiated. kits
[0105] [0105] A kit as used herein may comprise the compositions described herein together with some or all of the following: test reagents, buffers, probes and / or initiators, and sterile saline or other pharmaceutically acceptable suspension or emulsion base. In addition, kits may include instructional materials containing instructions (for example, protocols) for practicing the methods described here. highlighter
[0106] [0106] "Marker", as used herein, means a composition detectable by spectroscopy, photochemical, biochemical, immunochemical, chemical, or other physical device. For example, useful markers include * ºP, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities that may be detectable. A marker can be incorporated into nucleic acids and proteins at any position. pairing error
[0107] [0107] "Mismatch" means a nucleobase of a first nucleic acid that is unable to pair with a nucleobase in a position corresponding to a second nucleic acid. modified oligonucleotide
[0108] [0108] "Modified oligonucleotide", as used herein, means an oligonucleotide that has one or more modifications in relation to a naturally occurring internucleoside, terminal, sugar and / or nucleobase bond. modulation [01 09] "Modulation", as used herein, means a disturbance of function and / or activity and / or structure. In some modalities, modulation means an increase in gene expression. In some embodiments, modulation means a reduction in gene expression. mutant
[0110] [0110] "Mutant", as used herein, refers to a sequence in which at least a portion of the sequence's functionality has been lost, for example, changes in the sequence of a promoter or enhancer region will at least partially affect the expression of a coding sequence in an organism. As used herein, the term "mutation" refers to any change that eventually arises in the sequence of a nucleic acid sequence, for example, from deletion, addition, substitution, or rearrangement. The mutation can also affect one or more steps that the sequence is involved in. For example, a change in a DNA sequence can lead to the synthesis of an altered mRNA and / or an active, partially active or inactive protein.
[0111] [0111] "Nucleic acid sequence" or "oligonucleotide" or "polynucleotide", as used herein, means at least two nucleotides covalently linked together. The representation of a single filament also defines the sequence of the complementary filament. Thus, a nucleic acid further comprises the complementary strand of a single represented strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also comprises substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under strict hybridization conditions. Therefore, a nucleic acid also includes a probe that hybridizes under strict hybridization conditions.
[0112] [0112] Nucleic acids can have a single strand or two strands, or contain portions of the sequence with two strands or with a single strand. The nucleic acid can be DNA, genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, innosine na, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained with chemical synthesis methods or with recombinant methods.
[0113] [0113] A nucleic acid, in general, will contain phosphodiester bonds, although nucleic acid analogs that have at least one different bond may be included, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds and skeletons and nucleic acid-peptide bonds.
[0114] [0114] "Functionally linked", as used herein, may mean that the expression of a gene is under the control of a promoter to which it is spatially connected. A promoter can be positioned 5 '(upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately equal to the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, the variation of this distance can be adjusted without losing the function of the promoter.
[0115] [0115] "Promoter", as used herein, can mean a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further intensify expression and / or alter its spatial and / or temporal expression. A promoter can also comprise distal enhancer or repressive elements, which can be located thousands of base pairs away from the transcription start site. A promoter can be derived from sources such as viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively, or differentially in relation to the cell, tissue or organ in which the expression occurs or, in relation to the stage of development in which the expression occurs, or in response to external stimuli, such as physiological stresses, pathogens, metal ions, or inducing agents. Examples represented
[0116] [0116] “Recombinant host cells" refers to cells that have been transformed with vectors constructed using recombinant DNA techniques. Selectable marker
[0117] [0117] "Selectable marker", as used here, can mean any gene that confers a phenotype on a host cell, tissue, organ, callus or organism in which it is expressed to facilitate its identification and / or the selection of those that were transfected or transformed with a genetic construct. Representative examples of selectable markers include the ampicillin resistance gene (AmpR), tetracycline resistance gene (TcR), bacterial kanamycin resistance gene (KanR), zeocin resistance gene, the AURI-C gene that confers resistance to antibiotic aureobasidine A, the phosphinothricin resistance gene (Bar), neomycin phosphotransferase gene (nptll), hygromycin resistance gene, beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, gene encoding the green fluorescent protein (GFP) and the luciferase gene. In some modalities of this invention, a selectable marker can be produced from a modification of an endogenous gene, for example, the elimination of a chemokine receptor expressed and displayed on the surface of a cell when a mutation of this gene results in a reading frame shift mutation and can then be selected negatively with an antibody, or for example, a WB568L mutation in the smoke acetolactate synthase gene, which results in resistance to herbicides.
[0118] [0118] "Strict hybridization conditions", as used herein, means conditions under which a first nucleic acid sequence (eg probe) will hybridize to a second nucleic acid sequence (eg target), such as , in a complex mixture of nucleic acids. Strict conditions are dependent on the sequence and will be different in different circumstances. Strict conditions can be selected between about 5-10ºC below the thermal melting point (Tm) for the specific sequence in defined ionic resistance and pH. The Tm can be the temperature (under defined ionic resistance, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the equilibrium target sequence (as the target sequences are present in excess, in the Tm, 50% of the probes are occupied in equilibrium).
[0119] [0119] Strict conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, for example, sodium ion concentration of about 0.01-1.0 M (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30ºC for short probes (for example, about 10-50 nucleotides) and at least about 60ºC for long probes (for example, above about 50 nucleotides). Strict conditions can also be achieved with the addition of destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times the previous hybridization. Examples of strict hybridization conditions include the following: 50% formed, 5x SSC, and 1% SDS, incubating at 42ºC, or, 5x SSC, 1% SDS, incubating at 65ºC, with washing in 0.2x SSC , and 0.1% SDS at 65ºC.
[0120] [0120] "complementary", as used herein, means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99 % identical
[0121] [0121] "Substantially identical", as used herein, means that a first and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or in relation to nucleic acids, if the first sequence is substantially complementary to complement of the second sequence.
[0122] [0122] "Target nucleic acid" or "target sequence", as used herein, is any predetermined nucleic acid sequence in which it is desired to act, including, but not limited to, by coding or non-coding sequences, genes, exons or introns, regulatory sequences, intergenic sequences, synthetic sequences and intracellular parasitic sequences. In some embodiments, the target nucleic acid resides within a target cell, tissue, organ or organism. The target nucleic acid comprises a target site, which includes one or more nucleotides within the target sequence, which are modified to any degree with the methods and compositions disclosed herein. For example, the target site can comprise a nucleotide. For example, the target site can comprise 1-300 nucleotides. For example, the target site can comprise about 1-100 nucleotides. For example, the target site can comprise about 1-50 nucleotides. For example, the target site can comprise about 1-35 nucleotides. In some embodiments, a target nucleic acid may include more than one target site, which may be identical or different.
[0123] [0123] "Functional modification of the addressed gene" and "modification of the target gene" are any genetic techniques that result in permanent or temporary alteration of a target nucleic acid, including, but not limited to, deletion , insertion, mutation, substitution, nicking, methylation, acetylation, ligation, recombination, helical exhalation, chemical modification, marking, activation, inactivation and repression of one or more nucleotides in a target sequence . therapy
[0124] [0124] “Therapy”, as used here, means a method for the treatment of illnesses. In some modalities, therapy includes, among other things, chemotherapy, surgical resection, transplantation, and / or chemoembolization. transgenic organism
[0125] [0125] The term is intended for an organism that has one or more modification (s) of the target gene in its genome, introduced by the compositions and methods disclosed here. For example, the modification is selected from: insertion, mutation, substitution of one or more nucleotides, nicking, methylation, acetylation, binding, recombination, helical exhalation, chemical modification, marking, activation, inactivation and / or repression. The organism can be any type of organism, for example, human, animal, vegetable, and the like. transient expression
[0126] [0126] “Transient expression” or “transiently expressed”, as used here, can refer to the transcription, or translation of a nucleic acid supplied in a complete or partial organism, organ, tissue, callus or cell, differentiated or undifferentiated, said expression being limited due to the non-integration of the nucleic acid supplied to the stable nucleic acids of the organism, organ, tissue, callus or cell comprising the genome or organellar nucleic acids. Vectors for transient expression comprise linear or circular ssDNA, dsDNA or RNA, plasma
[0127] [0127] Treating ”or“ treating ”, as used herein, in reference to the protection of an individual against a certain condition may mean preventing, suppressing, suppressing, or eliminating the condition. Preventing the condition involves administering a composition described herein to an individual prior to the onset of the condition. Suppressing the condition involves administering the composition to an individual after the induction of the condition, but before its clinical appearance. Suppressing the condition involves administering the composition to an individual after the clinical appearance of the condition to reduce the condition or prevent its development. Eliminating the condition involves administering the composition to an individual after the clinical appearance of the condition so that the individual will no longer suffer from the condition. variant
[0128] [0128] "Variant", as used herein, in reference to a nucleic acid means (i) a portion of a cited nucleotide sequence; (ii) complementing a cited nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a mentioned nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under strict conditions to the aforementioned nucleic acid, its complement, or in a sequence substantially identical thereto. vector
[0129] [0129] "Vector" as used herein means a nucleic acid sequence used for the delivery of the nucleic acid. A vector can be used in this invention to cause genetic transformation, the expression of a protein, the transcription
[0130] [0130] As used herein, the term "wild type" sequence refers to a coding, non-coding or interface sequence that is an allelic form of the sequence that performs the natural or normal function for that sequence. Wild-type sequences include multiple allelic forms of a cognate sequence, for example, multiple alleles of a wild-type sequence can encode silent or conservative changes in the protein sequence that a coding sequence encodes.
[0131] [0131] According to some modalities, the composition comprising the programmable molecular complex that includes a fraction (polypeptide) of the protein, and a fraction of the nucleic acid, forms assemblies autonomously in vivo in a cell, organism, tissue, callus, organ or living part of those cited in the presence of a target nucleic acid sequence (s) to form programmed and active functional molecular complexes.
[0132] [0132] According to some modalities, the various programmed molecular complexes can be constructed to permanently or transiently modify an existing or imminent eukaryotic, prokaryotic, synthetic, intracellular or viral target sequence, such as those found in the genome, core,
[0133] [0133] According to some modalities, and without being bound by any theory or mechanism, the design of the programmable molecular complex is based on its ability to form autonomous assemblies, its ability to address a predefined intended sequence in a nucleic acid- target, and its ability to act on the target sequence in a predetermined manner. The components of the complex are modular and adjustable to suit 1) the particular types of molecular action required, 2) the target, and 3) the delivery method of the desired nucleic acid used for its in vivo expression. The methods and compositions of the present description have numerous advantages over other systems known in the art. For example, fractions of the complex protein are inactive as monomers, and only the correct spacing, in a limited range, of two SCNA oligonucleotides that bind to the target nucleic acid in a predetermined sequence, will result in positioning of the effector domains of the protein fractions in order to dimerize and be able to act specifically on the desired predetermined target site. This configuration, through which only dimers of programmed molecular complexes (ie, complex comprising a fraction of protein linked to SCNA, which is linked to the target nucleic acid), reduces or completely eliminates potential cleavage at site incorrect or non-specific, since the protein fraction itself does not bind to the target nucleic acid and does not act as a monomer.
[0134] [0134] According to some modalities, the active portion (functional domain) of the molecular complex is designed to be activated after the dimerization of the functional domain of the protein fraction only. The unscheduled protein component is designed to have low specific affinity for the nucleic acid sequence and the target site, or for that specific affinity to be virtually non-existent. Thus, although for all types of modifications, the expression of a single type of monomer of the protein fraction is necessary, for the minimal point modification functions, such as, for example, a domain-mediated point mutation of the nuclease, or alternatively, a methylation point mediated by a methylase domain, two SCNAs, designed to link sequences flanking the target site, must be present to interfere in the correct spacing of the proteins and allow mutual binding and dimerization. From an advantageous point of view, this increases the sequence specificity of the complex. In some modalities, for deletion and substitution editing functions, it may be necessary to cleave two different flanking sites in the region of interest to simultaneously. In this modality, even in this case, only one component of the exogenous protein needs to be expressed together with four SCNAs. When oligonucleotides are excluded, either by dilution or degradation, the expression of the unprogrammed protein has no affinity with the target nucleic acid and will cease to act on it (that is, in this case, the cleavage of the nucleic acid will cease. target).
[0135] [0135] According to some modalities, the protein fraction (polypeptide) can be expressed as separate polypeptides or as a contiguous protein (polypeptide). In some embodiments, the protein fraction (component) may have one or more identifiable domain (s), identifiable according to structure and / or function (utility). In some embodiments, a structural domain can have more than one utility domain, that is, a separable structural domain can have several functions. According to some modalities, the protein fraction may comprise one or more of the following structural and / or utility domains: a) an "effector domain" (functional domain), which can interact with and consequently affect the target nucleic acid; and / or b) a "binding domain", which can bind directly or indirectly to the SCNA in a specific way; and / or c) a "cell location domain"; and / or d) spacers or interdomain connectors; and any combination of those mentioned.
[0136] [0136] According to some modalities, the "Effector Domain" (here also called "Functional Domain"), interacts with the target nucleic acid after the assembly of the molecular complex and exerts the desired effect on the target. In some exemplary embodiments, this domain has an enzymatic or catalytic function, comprising a nucleic acid modification activity. In some embodiments, this domain can be derived from active domains derived from whole proteins, or from modified portions or portions of proteins of known function, such as a DNA binding protein, a nuclease, a methylase, a methylated DNA binding, a transcription factor, a chromatin modeling factor, a polymerase, a demethylase, an acetylase, a deacetylase, a kinase, a phosphatase, an integrase, a recombinase, a ligase, a topoisomerase , a gyrase and a helicase. In some embodiments, the functional domain can be constructed by fusing the amino acid sequence (s) of active domains derived from whole proteins, or from modified portions or portions of proteins of known function comprising a protein binding to DNA, a nuclease, a methylase, a methylated DNA binding factor, a transcription factor, a chromatin modeling factor, a polymerase, a demethylase, an acetylase, a deacetylase, a kinase, a phosphatase, an integrase , a recombinase, a ligase, a topoisomerase, a gyrase and a helicase. In some embodiments, for an effector domain that is or is derived from a nuclease, the nuclease DNA binding recognition domain can be removed. For example, when the effector domain is derived from a Fokl nuclease, the binding and recognition domains of the Fokl site are absent in the effector domain of the protein fraction. In some embodiments, the effector domain lacks a specific target nucleic acid binding site, that is, it cannot specifically bind to a specific target sequence.
[0137] [0137] According to some modalities, the "Binding domain" is designed to bind / attach, directly or indirectly, to the SCNA in a specific way (and in particular, to the SCNA recognition region). The connection / attachment between the link domain and the SCNA can be direct, or indirect through, for example, a modification in the SCNA. The attachments / ligation / unions between the binding domain and the SCNA allow the in vivo assembly of the SCNA with the protein fraction. In some embodiments, the binding domain is constructed by fusing the amino acid sequence of the protein fraction to amino acids that incorporate a domain that specifically binds to a nucleotide sequence or a chemical or biological element in the nucleic acid that provides specificity.
[0138] [0138] According to some modalities, a "cell localization domain" that can locate the protein fraction or programmed protein fraction or the complex assembled at a specific cell or subcellular location in a living cell, can optionally be part of the protein fraction. The cell localization domain can be constructed by fusing the amino acid sequence of the protein fraction to amino acids that incorporate a domain comprising a Nuclear Localization Sign (NLS); a mitochondrial leader sequence (MLS); a chloroplast leader sequence; and / or any sequences designed to transport or conduct or locate a protein in an organelle containing nucleic acid, a cell compartment or any subdivision of a cell. In some exemplary modalities, the organism is eukaryotic and the cell localization domain comprises a nuclear localization domain (NLS) that allows the protein to access the nucleus and the internal genomic DNA. The sequence of said NLS can comprise any positively charged sequence of functional NLS comprising, for example, the SV40NLS PKKKRKV sequence (SEQ ID NO: 3). In another exemplary fashion, this domain is formed by a leader sequence allowing the entry of the fraction of protein or nucleoprotein programmed into an organelle, enabling the desired modification of the organelle's DNA by the complex. In another exemplary modality, a sequence derived from the myxochondrial yeast Cox4p (MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID: 4)) or a sequence derived from the leading mitochondrial sequence of human malhydrate dehydrogenase (MLS) (MLSALARPASAALRRSFSTSAQ )) or derived from Arabidopsis lipoic acid synthase (NCBI Ref. Seg. ID: NP 179682.1 here designated as SEQ ID No.: 6: MHSRSALLYRFLRPASRCFSSSS) can be used to locate the complex in a mitochondrial matrix to modify the Mitochondrial DNA. One of the uses of this application would be to cure mitochondrial defects in maternal inheritance DNA in several eukaryotes, such as, Progressive External Chronic Ophthalmoplastic Syndrome in humans. Another example is to induce defects to cause male sterility in plants used in the production of hybrid plants. In one embodiment, the mitochondrial target is an ATPase and reconstitutes the function of the pcf locus in Petunia.
[0139] [0139] According to other modalities, several interdomain connectors or optional spacers are designed to allow the desired function of the complex acting as joints or molecular adapters. Many of these connectors can be seen by those skilled in the art. The connector choice can affect the specificity of the programmed molecular complex affecting the acid range
[0140] [0140] According to some modalities, the fraction of the nucleic acid of the molecular complex, here called "Specificity-promoting nucleic acid" (SCNA) or "programming nucleic acid" comprises one or more portions (regions) and functions. A portion (region) defines the target region to be attacked, and contains the specificity defining sequence. The specificity defining sequence in the SCNA defines its specificity to the target nucleic acid by base pairing. This pairing can form, for example, but not limited to: complete or partial double helix, complete or partial triple helix, D loops and branched shapes, which may be the result of hydrogen bonds or Hoogsteen or hydrogen bonds of your combinations. In some modalities, the specificity defining sequence is able to interact with the target nucleic acids, in the nearby regions, or flanking the target site. In some modalities, the SCNA's specificity-defining sequence does not bind / interact with the target site. In some embodiments, the specificity defining sequence can include any number of nucleotides. For example, the specification defining sequence
[0141] [0141] According to some modalities, a second portion of the SCNA, is the recognition region (portion), which is a region capable of | attaching / attaching / specifically recognizing the binding domain of the protein fraction. In some embodiments, this recognition region may be and / or include a modification or sequence of recognition of the binding domain (here also referred to as the SCNA nucleotide motif or the binding nucleotide sequence of the SCNA binding domain). The recognition region can be an integrated part or it can be linked (for example, covalently) to the specificity defining sequence, and consist of a sequence or a modification that allows the connection of the SCNA to the binding domain of the fraction of protect- Ína, as detailed above.
[0142] [0142] In some embodiments, SCNA consists of, but is not limited to, a molecule of the following types: single-stranded DNA, single-stranded RNA, double-stranded RNA, modified DNA, modified RNA , blocked nucleic acid (LNA), peptide-nucleic acid (PNA) and any combinations of those indicated. In some modalities, the SCNA may additionally include one or multiple modifications capable of enhancing stability, enhancing its specificity to the target, modifying its affinity for nucleic acids and / or enhancing its binding to the complex's binding domain. The modifications can be positioned at its 5 'end, at its 3' end, as spacers and / or internally in the SCNA. Exemplary modifications include, but are not limited to, Nucleotides, Biotin, Fluorescein, Amine Linkers, Oligopeptides, Aminoalyl, a Dye Molecule, Fluorophores, Digoxigenin, Acridite, Adenylation, Azide, NHS-Ester, Cholestyl-TEG, Alkaline, Biotin Photocleavable, Thiol, Dithiol, modified bases, phosphate, 2-Aminopurine, Trimer-20, 2,6-Diaminopurine, 5-Bromo-deoxyUridine, DeoxyUridine, inverted dT, didesoxy-nucleotides, 5-methyl deoxyCitidine, deoxyne , 5-nitroindole, 2-O-methyl RNA bases, Iso-dC, Iso-dG, Modified fluorine bases, Phosphorothioate bonds and the Agrobacterium VirD2 protein and parts of said VirD2 and VirD2 modifications.
[0143] [0143] According to some modalities, the SCNA may additionally include optional spacer sequences possibly useful in optimizing the molecular distances and degrees of freedom necessary to assemble the ligation domain and a target nucleic acid. In some embodiments, spacer sequences can be about 0-100 nucleotides in length. For example, the spacer can be about 0-6 nucleotides in length.
[0144] [0144] According to some modalities, SCNA can be produced chemically and / or biologically, in vitro and in vivo, and the modification can be previously synthesized or added after synthesis. In some exemplary modalities, SCNA is produced chemically and is formed by phosphorothioate-modified ssSDNA which is modified at one of its terminals through the connection of a Fluorescein C6 dye molecule. This SCNA is consequently delivered to a cell, (for example, with the use of particle bombardment, polyethylene glycol transfection, liposomes, viral particles, silicon carbide whiskers and / or electroporation) where both the protein component of the complex enters molecular, which comprises a Binding Domain comprising a ScFV single chain antibody capable of binding to the Fluorescein dye, thereby programming the molecular complex, and delivering / addressing the complex to the desired target nucleotide sequence. According to some modalities, SCNA does not bind / interact with the target site.
[0145] [0145] At this moment we refer to Figures 1A-B, which are schematic (out of scale) boards showing the elements / components of a programmable molecular complex, according to some modalities. The schematic boards (out of scale) of Figures 1A-B, show a molecule of a protein fraction programmable in the form of a monomer, and two molecules of specificity-promoting nucleic acids (SCNA). As Figures 1A-B show, the protein fraction is a polypeptide (a chain of amino acids) organized into several structural / functional domains: a binding domain (LD), a functional or effector domain (FD); an optional cell location domain (CLD) and an optional interdomain connector (s) (IDC), each defined by its function in the molecular complex. The function of the connection domain is to connect to the SCNA. The function of the effector domain is to interact with the target nucleic acid and structurally modify the target site and / or modify its function and / or the function of the entire target nucleic acid. The function of the optional cell localization domain is to locate the protein complex for the same cell or subcellular compartment as the target nucleic acid. The function of the optional interdomain connectors is to allow ideal molecular distances and degrees of freedom between the domains for the proper function of the complex. SCNAs are formed by a nucleic acid chain or modified nucleic acid chain (comb shape) and include a modification, preferably at one of its terminals (shown in Figure 1A as a black ellipse) to bind to the fraction of protein, or a sequence, (called an SCNA nucleotide motif, or binding nucleotide sequence of the binding domain or segment or recognition sequence of the binding domain, shown in Figure 1B, comb marked with an arrow), which can bind to the binding domain in the protein fraction. In the non-limiting example shown in Figures 1A-B, the specificity-determining portion of the SCNA has a single strand. In some
[0146] [0146] At this moment we refer to Figures 2A-B, which are schematic boards showing the assembly of the programmable molecular complex, according to some modalities. The schematic boards (out of scale) of Figures 2A-B demonstrate the way of assembling the components of the programmable molecular complex in a target nucleic acid. In the example shown in Figures 2A-B, two protein monomers bind to two different SCNAs, each having a different specificity determinant in its variable sequence region. These SCNAs pair with the bases and bind to homologous sequences predefined in a target nucleic acid (marked in the Figures as "target nucleic acid"). This base pairing can form a double or triple helix with the target nucleic acid, depending on whether the target has one or two strands (illustrated in these figures as dsDNA). Both SCNAs can connect to the same or opposite filaments as needed, at an optimized distance. SCNAs can bind to the Binding Domain of the protein through a modification in its terminal (Figure 2A) or through a nucleotide motif of the SCNA (Figure 2B). After assembly, the functional domain has its effect on the predetermined target site (marked "Target site") in the target nucleic acid.
[0147] [0147] At this point we refer to Figure 3, which demonstrates an example in a three-dimensional model of a molecular complex designed for the cleavage of a predefined nuclear dsDNA target sequence, according to some modalities. A fraction of programmed dimerized protein is shown in association with its target DsDNA (A, shown in part). Each monomer of the protein fraction is formed by a functional Domain derived from a subunit of the Fokl nuclease (B); a cell localization domain derived from SV40NLS (C); a binding Domain derived from an anti-fluorescein single-chain variable fragment antibody (anti-FAM ScFV, D) and an interdomain connector (E). Each Binding Domain (D) is shown linked to a specificity-enhancing nucleic acid, SCNA ssDNA (F, shown in part) through its modifying Fluorescein 6- carboxy molecule (G), which is covalently linked to the end of each SCNA . Cleavage sites (target site) expected from the target dsDNA (shown as spheres in the helix skeleton) are marked with arrows 300A-B. Each SCNA is represented here so as to form a partial triple helix that occupies the main groove of the flanking sequence of the target dsDNA.
[0148] [0148] At this moment we refer to Figures 4A-B, which are schematic drawings (out of scale) of the exemplary way of assembling the components of the programmable molecular complex in a target nucleic acid, according to some modalities. As the non-limiting examples shown in the
[0149] [0149] According to some modalities, the methods for delivering the SCNA to the organism or cell comprise several methods familiar to individuals skilled in the art and in general are ideal for the type of organism or cell used in the relevant circumstance. These may include the delivery of nucleic acid using biological methods of: infection with autonomously replicating vectors, transduction or infection of transgenic viruses, including the use of decontaminated or partial viruses, inoculation, delivery of Agrobacterium T-DNA , breeding, crossing, grafting, organelle transfer, chromosome transfer, cell fusion; chemical-mediated absorption methods of: using transfection agents, DEAE-Dextran, calcium phosphate, artificial lipids, dendrimers, polymers (PEG etc.), proteins / peptides, virus-like particles; the mechanical methods of: bombing, injection / microinjection, pressure, whiskers; and the electrical method of electroporation, and any method that alters the cellular plasma membrane, allowing nucleic acids to actively or passively penetrate the cell.
[0150] [0150] According to some modalities, the methods for delivering the nucleic acid that encodes the protein module to the organism or cell comprise numerous methods familiar to individuals skilled in the art and in general are ideal for the type of organism or cell used in the relevant circumstance. These may include the delivery of nucleic acid through crossing or enhancing an organism with a transgenic organism carrying the gene or through biological methods of: infection with autonomously replicating vectors, transduction or infection of transgenic viruses, including the use of uncontrolled viruses or partial, inoculation, delivery of Agrobacterium T-DNA, grafting, organelle transfer, chromosome transfer, cell fusion; the chemical-mediated absorption methods of: use of transfection agents, DEAE-Dextran, Phosphate
[0151] [0151] According to some modalities, the methods for delivering “donor DNA”, in the subgroup of demanding DNA uses that include gene insertion or gene replacement, comprise methods similar to those described for delivery of the nucleic acid encoding the protein. This DNA can be a single strand, two strands or partially two strands, linear or circular. This DNA can be supplied in a single vector or in several vectors, simultaneously or separately from the nucleic acid that encodes the protein component of the molecular complex and the specificity-providing programming nucleic acid. In this way, nucleic acids can be delivered from the choice of the appropriate delivery methods already mentioned, to a plant or part of a plant, to a plant, tissue or organ, such as an embryo, pollen, egg, before , stigma, complete flower, cotyledon, leaf, root, stem, petiole, to isolated plant cells, such as protoplasts, or tissue, callus, or cultured, differentiated or undifferentiated plant cells. In some embodiments, nucleic acids can be delivered to a fungus, including unicellular and multicellular fungi, and to a member of the animal kingdom, including invertebrates (such as arthropods and nematodes), vertebrates (such as birds, fish, mammals, reptiles, and amphibians) and parts of these organisms that include organs, organ culture, tissues, tissue culture, isolated cells, cell cultures, cell lines and stem cells, such as human embryonic stem cells or cells -human hematopoietic trunk.
[0152] [0152] At this point, we refer to Figure 5 which shows a schematic illustration showing the delivery options for the programmed molecular complex.
[0153] [0153] At this point we refer to Figure 6, which is a general scheme showing the delivery of the programmable molecular complex to a cell using an SCNA produced in vivo, according to some modalities. A nucleic acid molecule encoding the protein fraction is selected from the left column and delivered using applicable methods selected from the following three columns. SCNAs produced in vivo are encoded by a nucleic acid molecule supplied for that purpose and introduced into the cell using these same methods. The nucleic acid molecules encoding the protein fraction and / or the SCNA can be delivered separately or concurrently. In the cell, the nucleic acid encoding the SCNA expresses the SCNA through transcription or cleavage of the nucleic acid. If the delivered nucleic acid molecule is formed by dsDNA, it can first be transcribed into RNA via a designated promoter. If the delivered nucleic acid molecule is formed by ssDNA, it can first be complemented by dsDNA and then transcribed. If the delivered nucleic acid molecule is formed by RNA, such as a virus encoder or another autonomous replication vector, it can proceed through replication through a minus filament. Inside the cell, the nucleic acid encoding the protein is expressed through its in vivo translation from an RNA molecule produced in a manner similar to that described for SCNA. The translated protein can then be located in the desired cell subcompartment, according to its localization signal (if present). The nucleic acid molecules encoding the protein fraction and / or the nucleic acid molecules encoding the SCNAs can be delivered concurrently (at the same time) or separately, using identical or different delivery methods. Once the SCNA, protein fraction and target nucleic acid are colocalized within the cell, they can come together to form an active molecular dimeric complex. Donor DNA, if necessary, can also be delivered separately or simultaneously.
[0154] [0154] According to some modalities, the in vivo biological synthesis of SCNA can be carried out through several routes, such as, but not limited to: (a) the use of Agrobacterium to synthesize the nucleic acid and the binding fraction of the Binding Domain, in this example, VirD2, which also catalyzes its covalent binding. Agrobacterium then facilitates the transfer to a cell of a ssDNA | i-
[0155] [0155] At this point we refer to Figures 7A-B, which are schematic drawings (out of scale) showing non-limiting examples of the delivery of SCNA to a cell using a single-stranded DNA produced in Agrobacterium. Figure 7A shows a non-limiting example of the use of Agrobacterium for the production of SCNA ssDNA bound to a protein, VirD2, in vivo, at its 5 "end. As this example shows, the variable SCNA addressing sequence is inserted into a multiple cloning site (MCS) in a plasmid capable of replicating in Agrobacterium, Agrobacterium is then transformed with this plasmid. The Right Edge (RB) sequence of the Ti plasmid in the plasmid is cleaved and the ssDNA is VirD2 linked to bacteria.3 nucleotides of the RB sequence are left at the 5 'position of the sequence after cleavage, and 21 nucleotides of the Right Edge (RB) sequence of the Ti plasmid are left after cleavage at the 3' position of the sequence The LB sequence can further assist in the stabilization of the SCNA and in the evaluation of unwanted integration events The mutated form of Agrobacterium, (for example, lacking VirE1 or VirE2 or with partial VirD2 functionality) is useful in inhibiting integration events in desired. THE
[0156] [0156] At this point we refer to Figures 8A-B, which are schematic illustrations showing the delivery of SCNA to a cell using RNA SCNAs produced within the host cell, from a T-DNA delivered by Agrobacterium (Figure 8A) or from a nucleic acid delivered by an autonomously replicating vector, such as a virus, for example (Figure 8B). The RNA SCNAs shown in these figures include an SCNA-RNA motif (marked combs) that can bind to a corresponding RNA binding motif in the protein fraction binding domain. As shown in Figure 8A, the SCNA sequences are inserted into a multiple cloning site (MCS) on a plasmid capable of replicating in Agrobacterium and containing the appropriate eukaryotic promoters for the transcription of one or multiple RNA SCNAs in the infected cell. Figure 8B: The SCNA sequence (s) is / are inserted into the genome of a virus or an autonomous replication vector derived from a virus, each under the control of a subgenomic promoter (sg) for the transcription of one or multiple RNA SCNAs in the infected cell. In the non-limiting examples shown in Figures 8A-B, the
[0157] [0157] At this point, we refer to Figure 9, which shows a schematic illustration (out of scale) of a non-limiting example of a vector or delivery vehicle for the concomitant delivery of the composition comprising the components necessary for the assembly of a programmable molecular complex in a susceptible eukaryotic target cell in a single delivery event, according to some modalities. For the non-limiting example shown in Figure 9, the desired action is to replace an extension of the genomic DNA (the target nucleic acid) with a predetermined sequence, the "Donor cassette". Therefore, the domains of the protein fraction include: a functional Domain, derived from a nuclease and endowed with nucleic cleavage activity; a cell location domain, which is a nuclear location signal (NLS); and Binding domain capable of recognizing and binding to an RNA motif in SCNAs. In the example shown in Figure 9, a biological delivery system is used. Agrobacterium is transformed with a plasmid vector, such as plasmid (800), which contains various functional / structural sequences, such as, selectable bacterial marker, various origins of replication sites (origin of E. Coli, pSa Ori), LB sequence, promoter regions (designated as (P)), the sequence that expresses the protein fraction (comprising an initial ATG codon and a STOP codon within the frame), Terminator (T) site, multiple cassettes of SCNA transcription (shown as four SCNA transcription cassettes, each comprising a promoter and terminator sequences), a Donor cassette, and RB site. The plasmid vector (Transfected Agrobacterium) is then placed in contact with the organ cells.
[0158] [0158] According to some modalities, and as detailed above, changes / modifications in the addressed sequence include, for example, but are not limited to: permanent deletion, mutation, insertion of nucleic acids, and replacement of a sequence addressed by another nucleic acid sequence, knock-out, reading frame shift, or any change in the transcription or translation of a gene, its regulatory sequences, the regulatory genes of the gene of interest or its regulatory sequences in a regulatory chain of events.
[0159] [0159] At this point, we refer to Figure 10, which is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to create a mutation in a target nucleic acid, according to some modalities. As the non-limiting example shown in Figure 10 shows, the functional domain of the protein fraction is derived from a nuclease, and the target site mutation in the target nucleic acid is achieved by creating a dsDNA (DSB) break in Target nucleic acid at a predefined location. The SCNA programmed molecular complexes join together by pairing the SCNA base with a corresponding target sequence in the target nucleic acid. After assembling the complex components, the functional domain is dimerized and the nuclease is activated, cleaving the target site, which is located, in this example, at or near the midpoint, between the two SCNA molecules, thus creating a DSB (for example, the DSB may have 5 'overhangs on nucleotide 4, such as those created by the restriction enzyme Fokl). Mechanisms of cellular repair by non-homologous recombination (NHEJ) try to repair the DSB and in this attempt they can: 1) make a perfect connection while the complex can continue cleaving the same sequence again several times in the mutation until component depletion of the complex, 2) adding one or multiple nucleotides, thereby expanding the distance between the SCNAs and eliminating dimerization of the functional domain, thereby ending the action of the complex, or 3) removing one or multiple nucleotides (figure "pacman"), thereby reducing the distance between the SCNAs and eliminating the dimming of the functional domain, thereby ending the action of the complex. When either option 2 or 3 occurs inside the cell, a change is achieved.
[0160] [0160] At this point we refer to Figure 11, which is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to insert one or multiple nucleotides into a target nucleic acid using a Donor nucleic acid provided, according to some modalities. As the non-limiting example shown in Figure 11 shows, the functional domain of the protein fraction is derived from a nuclease, and a breakdown of dsDNA (DSB) in the target nucleic acid at a predefined location (target site) assists the process of homologous recombination (HR). The programmed molecular complexes of SCNA come together
[0161] [0161] At this point we refer to Figure 12, which is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to replace, insert and / or delete one or multiple nucleotides in a nucleic acid -Target using a donor nucleic acid provided, according to some modalities. As the non-limiting example shown in Figure 12 shows, the functional domain of the protein fraction is derived from a nuclease, and a breakdown of dsDNA (DSB) in the Target nucleic acid at a predefined location (target site) assists the process of homologous recombination (HR). The programmed molecular complexes of SCNA come together autonomously by pairing the base of the SCNA with a predetermined target sequence. After assembling the components of the complex, the functional domain is dimerized and the nuclease is activated,
[0162] [0162] At this point we refer to Figure 13, which is a schematic illustration (out of scale) demonstrating the use of a molecular complex programmed to create the deletion of one or multiple consecutive nucleotides from a nucleic acid- according to some modalities. As the non-limiting example shown in Figure 13 shows, the functional domain of the protein fraction is derived from a nuclease, and the deletion is achieved by creating two dsDNA breaks (DSBs) in the Target nucleic acid at two predefined locations. The programmed molecular complexes of SCNA come together autonomously by pairing the SCNA base with corresponding target sequences. After assembling the components of the complex, the functional domains are dimerized and the nucleases are activated, cleaving the target nucleic acid at the target site, which can be located at or near the midpoint between each pair of SCNA molecules creating DSBs. The simultaneous or sequential cleavage of both sites is
[0163] [0163] At this point we refer to Figure 14, which is a schematic illustration demonstrating the use of a molecular complex programmed to replace one or multiple nucleotides in a Target nucleic acid using a donor nucleic acid provided, according to some modalities. As the non-limiting example shown in Figure 13 shows, the functional domain of the protein fraction is derived from a nuclease, and replacement is achieved by creating two dsDNA breaks (DSBs) in the Target nucleic acid at two predefined locations ( targets), creating a deletion, and providing a linear or linearized donor DNA to fill the space. The programmed molecular complexes of SCNA come together autonomously by the base pairing of the SCNA with the corresponding target sequences. After assembling the components of the complex, the functional domains are dimerized and the nucleases are activated, cleaving the target at or near the midpoint between each pair of SCNA molecules, thus creating the DSBs. The simultaneous or sequential cleavage of the two sites essentially eliminates,
[0164] [0164] According to certain embodiments, the compositions and methods of the present invention can be used to replace any genomic sequence with a homologous, non-identical sequence. For example, a mutant genomic sequence can be replaced by its wild-type counterpart, thus providing methods for treating, for example, genetic disease, hereditary disorders, cancer, and autoimmune diseases. Likewise, an allele of a gene can be replaced by a different allele using the methods disclosed herein. Examples of genetic diseases include, but are not limited to, achondroplasia, achromatopsia, acid maltase deficiency, acquired immunodeficiencies, adenosine deaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardi syndrome, alpha-l antitrypsin deficiency, alpha thalassemia, insensitivity syndrome in erogenous zones, apert syndrome, arrhythmogenic right ventricular dysplasia, ataxia telangiectasia, barth syndrome, beta thalassemia, blue nevus syndrome (blue rubber bleb nevus), canavan disease, chronic granulomatous diseases (CGD ), cattle meow syndrome, cystic fibrosis, dercum disease, ectodermal dysplasia, Fanconi anemia, progressive ossifying fibrodysplasia, fragile X syndrome, galactosemia, Gaucher disease, generalized gangliosidosis (eg GM1), hemochromatosis, hemoglobinopathies (eg sickle cell anemia, the hemoglobin C mutation in the 6.sup.th beta-globin codon, thalassemia alfa, thalassemia be ta), hemophilia, Huntington's disease, Hurler's syndrome, hypophosphatasia, Klinefleter's syndrome, Krabbes's disease, Langer-Giedion's syndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920), leukodystrophy, long QT syndrome , lysosomal storage diseases (eg Gaucher disease, GM! 1, Fabry disease and Tay-Sachs disease), Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (eg Hunter disease, Hurler disease ), nail-patella syndrome, nephrogenic insipid diabetes, neurofibromatosis, Neimann-Pick disease, imperfect osteogenesis, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, thrombocytopenia syndrome with absence of radio (TAR), Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner syndrome, urea cycle disorders, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, Wiskott-Aldrich syndrome, linked lymphoproliferative syndrome to the X chromosome (XLP, OMIM No. 308240).
[0165] [0165] The following examples are presented in order to further illustrate some embodiments of the invention. However, they should not be
[0166] [0166] This example describes a bioassay suitable for testing and optimizing permutations in the design and use of the programmable molecular complex, for example, to test its activity on different organisms or cells, different delivery methods, and to test the mutation editing functions , substitution, deletion and insertion.
[0167] [0167] The experiments shown in the examples below serve to detect gene addressing and specific cleavage by a programmable molecular complex composition, which includes a modified nuclease as the protein fraction's effector domain.
[0168] [0168] Visual reporter systems are used based on the repair of a STOP codon that is inserted into the reporter coding sequence. The reporter in these examples is the green fluorescent protein (GFP). When addressed, breaks in the double filament (DSB) formed by the activated complex are repaired, (presumably through NHEJ as illustrated in Figure 10), eliminating the STOP codon and recovering GFP activity. Thus, this assay can satisfactorily indicate the efficiency of gene addressing. This test is also known as the “STOP GFP” test.
[0169] [0169] This visual assay is designed to target the plasmid or genomic DNA in vivo. In the examples below, a protoplast-based bioassay of Arabidopsis is used. In the described bioassay, the aforementioned reporter systems are delivered to the protoplasts in a plasmid, co-delivered with the plasmid expressing the protein fraction of the molecular complex in vivo and co-delivered with a modified ssDNA Specificity-Enhancing Nucleic Acids (SCNA) pair , in this example, with a terminal (NHS-Ester -) - Digoxigenin (DIG). A second modification to protect the exonuclease, (phosphorothioate), is added at the opposite end (here marked with an asterisk). The plasmid vectors used herein comprise plant promoters. Protein sequence and properties
[0170] [0170] The molecular complex designed for this application consists of two sequences of homologous nucleic acids for specificity determination (SCNAs) and a chimeric component of the protein containing a nuclease that binds to SCNAs in vivo. The resulting cleavage of the predetermined target site (STOP codon) of the target nucleic acid (GFP coding sequence) results in its desired mutation, through endogenous processes. The programmable molecular complex in this example consists of 2 identical monomers of a fraction of protein and two different SCNA molecules (as schematically illustrated in Figures 1A and 2A). In this example, the protein fraction contains an amino acid sequence modified from a Fokl nuclease domain as a functional domain; an amino acid sequence adapted from anti-DIG single-chain variable fragment (scFv) immunoglobulin (Digoxigenin) (DIG-ScFv) similar to the one described (Huston et. al, 1988) as Binding Domain; an SV40NLS (SEQ ID No.: 3, PKKKRKV) as a nuclear location domain and a -15Á interdomain connector (SEQ ID No.: 7, GGSGG). The nucleic acid sequence encoding the protein fraction is inserted into suitable expression vectors (vectors based on pUC (pSAT)), including a NOS or 358 promoter.
[0171] [0171] The in vivo link between the specificity-promoting nucleic acid and the protein fraction binding domain, in this example, is the result of a non-covalent interaction that can be described as an antigen-antibody interaction; single chain antigen-antibody; antibody or hapten-single chain antibody interaction.
[0172] [0172] In this example, the modification at the nucleic acid end of the SCNA is an NHS-Ester-linked Digoxigenin (DIG) that is attached to the 5 'or 3' position of the SCNA oligonucleotide.
[0173] [0173] The amino acid sequence (one-letter code) of the protein fraction of the molecular complex (sequence of the NLS-FokIl nuclease with Digoxigenin ScFv) was designed as in SEQ ID: 12, and is encoded by the sequence as described described in SEQ ID No.: 13.
[0174] [0174] The length of the complementary target base pairing oligonucleotide SCNA is preferably at least 18 bases. The SCNA can also contain a small number (for example, 1-6, in one example, 6, in another example, 2) of unaddressed base pairing nucleotides (N's) of any sequence composition that serves as a spacer between the DIG-NHS terminal modifier and complementary target nucleotides. As detailed above, as histones occupy less significant DNA grooves in chromosomal DNA, there may be some restrictions on SCNA spacing. In this way, SCNAs are preferably designed to fit into the main groove of the target DNA by modulating the distance between the SCNAs, to allow an orientation of the target helix that allows the binding domains of a dimerized programmable molecular complex to be linked . The choice of the interdomain connector between the globular functional domain and the connection domain (in the example shown here is GSLEGGSGG (SEQ ID: 14)) also influences the ideal SCNA distance, as it restricts the movement in the “ articulation ”between these two domains. The addition of unaddressed base pairing nucleotides (“N's”) alters the distance between the SCNAs and the direction of rotation in the target helix, as it alters the SCNA's flexibility in relation to the protein and helix. These unpaired nucleotides are not restricted to the main groove of the target DNA.
[0175] [0175] The results of spatial measurements obtained with three-dimensional computerized models for the anti-DIG-ScFv-NHS-Ester-DIG system with the / inker of the interdomain GSLEGGSGG (SEQ ID: 14), as shown in this example, showed that the expected ideal distance between SCNAs, in the presence of 2 N's in the SCNA, corresponds to about 23-26 nucleotides. It is expected that the cleavage will occur about +2 nucleotides to the left and right of the 11th, 12th or 13th nucleotide, counting from the last nucleotide that hybridizes with the SCNA on both sides, taking into account the protrusion 5 '(overhang) of base 4 created by cleavage of dsDNA by the dimerized construct. This criterion suggests that, if the addressed sequence is, for this example with 24 nucleotides:
[0176] [0176] AAMAAAAAAA VV XKKKKXKVYYYYYYYCCCCCCOCCCC, where Y + X represents the number of nucleotides between the SCNA base pairing sites, so the projected SCNAs pair with areas A and C and the resulting cleavage in the DSB is in or adjacent to the area X. SCNAs can be complementary to the sense and antisense filaments, however, they are chosen so that the preferred pairing occurs with the sense (not transcribed) sequence. Both SCNAs can pair with the same filament, since the position of the protein fraction is located at the “close end” of the SCNA as defined by modification 5 'or 3' of the primer that is at the “close end” (as illustrated in Figure 2A). D Optimization of the distance between SCNAs, as well as the preferred filament, are one of the several criteria tested in this bioassay.
[0177] [0177] Target nucleic acid (GFP coding sequence), containing a target site (PARADA codon, (TAG)) includes the nucleotide sequence described in SEQ ID: 15 ("'STOP-GFP"), where the TAG stop codon is located at nucleotide 878:
[0178] [0178] The mCherry donor for examples 1B and 1C includes a coded sequence
[0179] [0179] The following target site sequence is addressed in examples 1A to 1C: Examples 1A-C “first target sequence”:
[0180] [0180] GTOGACAACTAGTCCAGATCOT (SEQ ID NO: 17) SCNA sequences
[0181] [0181] The modification symbols are: Phosphorothioate bonds = *; 5 'DIG = / SDigN /; 3'DIG = / 3DigN /).
[0182] [0182] Paired SCNA combinations tested for “first target” 1A- 1C: SCNA Direction:
[0183] [0183] GFP 918 SR1: / SDI9NNNNNNNNGTGTCCAAGGGCGAGGAGCTG * T; (nucleic acids are designated here only as SEQ ID No.: 18)
[0184] [0184] GFP 896 —SL1: T! -TTACGAACGATAGCCATGGCCNNNNNN / 3DigN / (nucleic acids are designated here only as SEQ ID NO: 19)
[0185] [0185] A second sense paired combination, employing a target gap of 24bp and a shorter SCNA linker according to the prediction results:
[0186] [0186] GFP 920 SR1: / SDIAONNNNGTCCAAGGGCGAGGAGCTGTT '* C (nucleic acids are designated here only as SEQ ID NO: 20)
[0187] [0187] GFP 895 SL1: A * TTTACGAACGATAGCCATGGCNN / 3DigN / (nucleic acids are designated here only as SEQ ID NO: 21) Anti-sense SCNA:
[0188] [0188] GFP 918 ASR1: CYAGCTCCTCGCCCTTGGAGACNNNNNN / 3DIGN / (nucleic acids are designated here only as SEQ ID NO: 22)
[0189] [0189] GFP 896 ASL1: / SDIGNINNNNNNGGCCATGGCTATCGTTCGTA * A (nucleic acids are designated here only as SEQ ID NO: 23)
[0190] [0190] A second antisense paired combination, employing a 24bp target gap and a shorter SCNA / inker according to the prediction results
[0191] [0191] GFP 920 ASR1: G * AACAGCTCCTCGCCCTTGGACNN / 3DIGN / (nucleic acids are designated here only as SEQ ID NO: 24)
[0192] [0192] GFP 895 ASL1: / SDIGNANNGCCATGGCTATCGTTCGTAAA * T (nucleic acids are designated here only as SEQ ID No.: 25) Combinations of sense and antisense pairs:
[0193] [0193] GFP 918 SR1: / SDIgN / NNNNNNGTGTCCAAGGGCGAGGAGCTG * T (nucleic acids are designated here only as SEQ ID No.: 18)
[0194] [0194] GFP. 896 ASL1: / SDIGNINNNNNNGGCCATGGCTATCGTTCGTA * A (nucleic acids are designated here only as SEQ ID NO: 23)
[0195] [0195] A second antisense paired combination, employing a 24bp target gap and a shorter SCNA / inker according to the prediction results:
[0196] [0196] GFP 920 SR1: / S5DI0NNNNGTCCAAGGGCGAGGAGCTGTT * C / (nucleic acids are designated here only as SEQ ID No.: 20)
[0197] [0197] GFP 895 ASL1: / SDIGNANNGCCATGGCTATCGTTCGTAAA * T (nucleic acids are designated here only as SEQ ID No.: 25)
[0198] [0198] GFP 918 ASR1: C'AGCTCCTEGCCOTTGGAGACNNNNNN / 3DIGN / (nucleic acids are designated here only as SEQ ID NO: 22)
[0199] [0199] GFP 896 —SL1: T-TTACGAACGATAGCCATGGCCNNNNNN / 3DigN / (nucleic acids are designated here only as SEQ ID NO: 19)
[0200] [0200] A second antisense paired combination, employing a 24bp target gap and a shorter SCNA linker according to the prediction results:
[0201] [0201] GFP 920 SL1: A * TTTACGAACGATAGCCATGGCNN / 3DigN / (nucleic acids are designated here only as SEQ ID No.: 21)
[0202] [0202] GFP 895 ASR1: G'YAACAGCTCCTCGCCCTTGGACNN / 3DIGN / (nucleic acids are designated here only as SEQ ID NO: 24)
[0203] [0203] “First target” for example, 1C is identical to target 1h and 1B.
[0204] [0204] “Second target” eg 1C: GACTCTAAGCTTGGGTCTAGA (SEQ ID: 26)
[0205] [0205] SCNAs for example 1C:
[0206] [0206] A combination, using a 24bp target gap and a short SCNA / inker: Direction:
[0207] [0207] GFP 1658 SR: / SDIGNINNTCCGCAAAAATCACCAGTCTC * T (nucleic acids are designated here only as SEQ ID NO: 27)
[0208] [0208] GFP 1633 SL: G * CATGGACGAGCTGTACAAGTCNN / 3DIGN / (nucleic acids are designated here only as SEQ ID: 28) Anti-sense:
[0209] [0209] GFP 1658 ASR: A * GAGACTGGTGATTTTTGCGGANN / 3DIGN / (nucleic acids are designated here only as SEQ ID NO: 29)
[0210] [0210] GFP 1633 ASL: / SDIGN / NNGACTTGTACAGCTCGTCCATG'C (nucleic acids are designated here only as SEQ ID NO: 30)
[0211] [0211] As with the “first target” 1A-C SCNAs, these four “second target” SCNAs in example 1C can be paired using a “left” (L) and a “right” (R) SCNA list above.
[0212] [0212] Bioassay configuration: The preparation of the Arabidopsis protoplast is based on Wu et. al. (Wu et. Al., 2009):
[0213] [0213] Plant Material: Arabidopsis grown under an ideal daylight regime of 16 hours (150microEinstein: m-2: s-1) at 22ºC.
[0214] [0214] Leaves: plants with 3 to 5 weeks of life (W -2cm L -5cm).
[0215] [0215] Enzyme solution: 1% cellulase, 0.25% Macerozyme, 0.4M Mannitol, 10mM CaCl2, KCl2OmM, 0.1% BSA, 20mM MES pH5.7. Heating at 50-55 ºC for 10 minutes to inactivate the proteases and then filter. Use 10ml / 7-10 dry peeled leaves (1-5gr) / plate.
[0216] [0216] Modified W5 solution: 154mM NaCl, CaCl2125mMM, KCISMM, 5mM Glucose, 2mM MES pH5.7. Wash twice with 25ml / plate, + twice 3ml for transfection bath + 1ml resuspension
[0217] [0217] Modified MMg solution: (Resuspension solution) 0.4 M Mannitol, MgCl215mM, MES 4mM pH5.7.
[0218] [0218] Modified TEAMP transfection buffer (PEG solution): 40% PEG MW 4000, CaCl2 0.1M, Mannitol 0.2M volume = 1: 1 of 200 protoplasmic microliter in MMg + volume of DNA
[0219] [0219] BSA: 1% BSA Working protocol:
[0220] [0220] Preheat in a water bath to 50-55 ºC, cool in a swing-out centrifuge, freeze W5 and MMg, and cut the tips.
[0221] [0221] Prepare plates covered with fresh BSA (1.25ml 1% BSA / well in water, incubate on the bench until ready)
[0222] [0222] Prepare 10ml fresh enzyme solution / treatment.
[0223] [0223] Collect 7-10 leaves, they must not be damp. 10 sheets should yield -4-5 transformations.
[0224] [0224] Cover the upper epidermis with Time-tape, the lower with Magic tape. Easier without gloves. It is easier to peel if the petiole is adhered only to the time-tape.
[0225] [0225] Filter in 0.22um 10ml of fresh enzyme solution in each Petri dish
[0226] [0226] Peel and discard the Magic tape. Transfer the side of the Time-tape to the Petri dish
[0227] [0227] Shake gently on a platform shaker at 40 rpm 20-60 min in the light until the protoplasts are released (check empirically)
[0228] [0228] Centrifuge in 50ml tubes 100 x g 3 min in swing-out rotor
[0229] [0229] Wash twice with 25ml of cold W5 solution.
[0230] [0230] Place on ice for 30 min, make contact during this time on the hemo-cytometer using a light microscope
[0231] [0231] Centrifuge and resuspend in MMg solution up to 2-5 x10º5 cells / ml (about 1 ml).
[0232] [0232] Produce fresh PEG solution for transfection in a 2ml tube!
[0233] [0233] Remove BSA from 6-well plates and dry
[0234] [0234] Mix -5 x 10% protoplasts (2 x 1074 -1 x 10º5) in 0.2 ml MMg solution with a mixture of target plasmid DNA, plasmid DNA that expresses the Fraction of protein and ssDNA of SCNAs until the total of 30-40 micrograms at RT in 15 ml round bottom tubes (cap).
[0235] [0235] Add equal volume (0.2ml protoplasts + midiprep vol.) Of fresh PEG solution
[0236] [0236] Incubate at room temperature for 5 min
[0237] [0237] Wash by slowly adding 3ml! of W5 solution, 1ml at a time, and mixing
[0238] [0238] Centrifuge at 100 x g in swing-out for 1 min
[0239] [0239] Repeat washing and pelletizing
[0240] [0240] Resuspend in 1ml! W5 solution
[0241] [0241] Pour into BSA-coated plates
[0242] [0242] Cultivate the protoplasts in the ideal daylight of 16 hours (15O0microEinstein: m / -2: s "-1) at 22ºC, replacing the medium as needed.
[0243] [0243] Protoplasts suspended in W5 solution are evaluated in terms of activity
[0244] [0244] In this example, cleavage of the target results in a Double Filament Breakdown (DSB) in the target DNA of the plasmid. This DSB is designed to be created at the PARADA codon site, which is digested and repaired by the NHEJ repair mechanism as described in the example illustration in Figure 10 (mutation). NHEJ is prone to mutations, and some of these mutations can eliminate the STOP codon and restore an open reading frame resulting in an active GFP open reading frame (ORF). The GFP is then detected by means of a microscope or flow cytometer (FACS), allowing to measure the efficiency of the system and the comparison between variables for its improvement.
[0245] [0245] During the addressing of a STOP-GFP transgene previously and stably introduced into the Arabidopsis genome (instead of a plasmid), plants with their modified genome can be regenerated from GFP-expressing protoplasts.
[0246] [0246] Like example 1A, the GFP stop codon sequence within the frame is addressed with the programmed molecular complex. In this application a donor linear dsDNA is added, comprising a mCherry reporter gene without a promoter and without a terminator containing only CDS. Following the described transfection, protoplasts that express mCherry are detected by red fluorescence through microscopy or flow cytometer (FACS), allowing to measure the efficiency of the system and the comparison between variables for its improvement. Excitation and emission of mCherry are 561nm and filter 610/20. Since the donor DNA contains a mCherry without a promoter, its activity can be obtained by capturing the promoter. In this way, the addressed GFP cassette is cleaved to form a DSB where any linear DNA can be linked. As an excess of linear dsDNA from CDS mCherry is supplied, it is captured in the DSB, causing, in some cases, translation in the framework of the mCherry protein. Plasmids addressed with that specific mCherry insert in the GFP addressed sequence are further analyzed by PCR with the following primers: one binding to the DNA sequence of the target plasmid, and one binding to the inserted DNA:
[0247] [0247] 35SF: CTATCCTTCGCAAGACCCTTCC (SEQ ID: 31)
[0248] [0248] mMCherryR: TIATOTTGTACAGCTCGTCCAT (SEQ ID: 32)
[0249] [0249] Similarly, resistance to bacterial antibiotics (NPT-Il coding cassette, without an origin of replication) is given to protoplasts as linear dsDNA. This DNA is inserted in place of mCherry CDS from examples 1B and 1C, and evaluated by extracting the total DNA from the protoplasts, transforming the plasmids that include DNA with or without inserts in E. Coli, and culturing them in a medium containing kanamycin. Resistant bacteria have plasmids that capture the NPT-Il cassette. To assess the specificity of insertion at the predetermined GFP target site, the GFP target site is amplified by PCR with primers spanning the expected insertion site. The specific insertion causes a significant variation in the size of the agarose gel PCR product. The efficiency of the insertion is calculated by dividing the number of colonies resistant to kanamycin by the number of colonies resistant to ampicillin (Resistance to ampicillin is encoded in the target plasmid) in an experiment with duplicate plates. Specificity is calculated by repeating the experiment with the omission or substitution of components of the programmable molecular complex (for example, GFP addressing SCNAs) and comparing to the unmodified experiments.
[0250] [0250] In this example, the GFP coding sequence is replaced by mCherry CDS via endogenous NHEJ. To exclude a large section of the target DNA with the NHEJ strategy, two DSBs are created. To address the start and end of GFP CDS, two groups of SCNAs are used together with the linear dsDNA donor mCherry. As the donor DNA contains mCherry without a promoter, its activity can be obtained by capturing the promoter. Therefore, the addressed GFP cassette can capture mCherry CDS. MCherry is analyzed by FACS or microscope with excitation and the emission detected at 561nm and filter 610 / 20s, respectively.
[0251] [0251] mCherry positive protoplasts are classified by FACS and subsequently subjected to DNA extraction, direct transformation of plasmids that include total DNA into E. Coli, growth in medium containing antibiotic, and performing two PCR reactions colony in each bacterial colony with two sets of primer:
[0252] [0252] 35SF: CTATCCTTCGCAAGACCCTTCC (SEQ ID No.: 31)
[0253] [0253] mMCherryR: TTITATOTTGTACAGCTCGTCCAT (SEQ ID: 32)
[0254] [0254] and
[0255] [0255] 385S-T-R-SEQ: CCCTATAAGAACCCTAATTCCC (SEQ ID No.: 33)
[0256] [0256] mCherryF: ATGGTGAGCAAGGGCGAGGA (SEQ ID NO: 34)
[0257] [0257] Colonies that produce an amplification product in both PCR reactions contain a plasmid that was addressed in the Arabidopsis protoplasts to produce a replacement event correctly oriented through the NHEJ repair pathway, and that afterwards undergo sequencing for verification purposes.
[0258] [0258] During the addressing of a GFP transgene previously and steadily introduced into the Arabidopsis genome (instead of a plasmid), the transformation
[0259] [0259] IPK1 addressing in corn for knockout.
[0260] [0260] The IPK1 gene encodes inositol-1,3,4,5,6-pentakisphosphate 2-kinase, which is involved in phytate biosynthesis in corn seeds. Phytate, when supplied to non-ruminant animals, is an anti-nutritional component that contributes to environmental pollution by phosphorus. E IPK1 addressing can reduce the seed's phosphorus content by 75%. There are two parallel IPK genes in Zea mays sharing 98% sequence identity in the corn genome. In this example, the IPK1 sequence based on Genbank Access No. EF447274 is addressed.
[0261] [0261] In exon 2 of IPK1: TICTCAAGTCATGAGCAACTOC (SEQ ID NO: 35)
[0262] [0262] Protein sequence and properties
[0263] [0263] The resulting IPK1 cleavage of the target site predetermined by the programmed molecular complex, results in its mutation or the insertion of a donor DNA in the DSB created by the programmed complex, as desired, aided by endogenous processes. In this case, the programmable molecular complex consists of 2 identical monomers of a protein fraction and two different SCNA molecules. In this example, the protein fraction is identical to that in example 1.
[0264] [0264] In this example, the modification at the nucleic acid end of the SCNA is an NHS-Ester-linked Digoxigenin (DIG) which is attached to the 5 'or 3' position of the oligonucleotide.
[0265] [0265] The logical design of the SCNA essentially corresponds to that described in Example 1. The length of the complementary target base pairing oligonucleotide preferably has at least 18 bases. The SCNA can also contain a small number (for example, 1-6, in one example, 6, in another example, 2) of unaddressed base pairing nucleotides (N's) of any sequence composition that serves as a spacer between the DIG-NHS terminal modifier and complementary target nucleotides.
[0266] [0266] Combinations of the following SCNAs "R" and "L" employing a target gap with 21bp are tested:
[0267] [0267] IPKI-SR-1710: / SDIGNINNNNNNCTGTGGGGCCATATCCCAGAA * C (nucleic acids are designated here only as SEQ ID NO: 36)
[0268] [0268] IPK1-SL-1688 :! GYCGGGCACCGAGTTGTATTGTANNNNNN / 3DIGN /. (nucleic acids are designated here only as SEQ ID No.: 37)
[0269] [0269] IPKI-ASR-1710: G * TTOCTGGGATATGGCCCCACAGNNNNNN / 3DIGN / (nucleic acids are designated here only as SEQ ID No.: 38)
[0270] [0270] IPK1-ASL-1688: / SDIGN / NNNNNNTACAATACAACTCGGTGCCCG * C (nucleic acids are designated here only as SEQ ID No.: 39)
[0271] [0271] A second combination group of paired "R" and "L" SCNAs, using a target gap of 24bp and a shorter SCNA linker according to the prediction results:
[0272] [0272] IPK1-SR-1712: / SDIAONNNNGTGGGGCCATATCCCAGAAC * T (nucleic acids are designated here only as SEQ ID: 40)
[0273] [0273] IPK1-SL-1687: A * GCGGGCACCGAGTTGTATTGTNN / 3DigN / (nucleic acids are designated here only as SEQ ID No.: 41)
[0274] [0274] IPK1-ASL-1687: / SDIONNNNACAATACAACTCGGTGCCCGC * T (nucleic acids are designated here only as SEQ ID No.: 42)
[0275] [0275] IPKI-ASR-1712: A * GTTCTGGGATATGGCCCCACNN / 3DigN / (nucleic acids are designated here only as SEQ ID No.: 43)
[0276] [0276] SCNAs comprise modified ssDNA. The modification symbols are: Phosphorothioate bonds = *; 5 'DIG = / SDigN /; 3 DIG = / 3DigN /.
[0277] [0277] In this experiment, the genomic DSB in corn plants and the specific integration of the GFP sequence into the IPK1 gene forming a knockout mutation and GFP expression in the / ocus IPK1 are tested. The programmed molecular complex forms the genomic DSB in the IPK1 sequence, initiating the integration of donor DNA into the IPK1 sequence through homologous recombination.
[0278] [0278] This example, 2A, is performed on corn protoplasts that are analyzed by FACS in terms of GFP activity.
[0279] [0279] A transient expression assay using corn mesophile protoplasts (Sheen, 2001) is used with electroporation-induced nucleic acid delivery in addition to or as an alternative to a polybrene-induced delivery protocol:
[0280] [0280] Transfection based on (Antonelli & Stadler, 1989):
[0281] [0281] Isolated fresh protoplasts (about 2x106) are incubated for about 6 to 12 hours with about 20-50 micrograms of transfection DNA comprising modified ssDNA SCNAs, a plasmid encoding the protein fraction, Donor DNA (where applicable ), and 30 micrograms of Polybene for policing (hexadimetrine bromide). At the end of the incubation period, the transfection mixture is diluted with the addition of growth medium and the cells are then incubated for approximately another 30 hours before being evaluated for transient gene expression:
[0282] [0282] Transfected corn protoplasts suspended in MS2D8M solution are analyzed by flow cytometer using fluorescence activated cell separation analysis (FACS), 3 days after transfection with Polybrene. GFP is detected by excitation at 488nm with emission detected by a 530/30 filter. Threshold and compensation factors are defined to exclude all false positives. FACS is used for separate addressed cells for further analysis.
[0283] [0283] The protoplasts are subjected to analysis through the extraction of genetic DNA
[0284] [0284] Initiator 1F: GAGCTAGATAGCAGATGCAGAT (SEQ ID: 44)
[0285] [0285] Initiator 2R: CTOCAGAAAATCCCTAGAAAÇCA (SEQ ID: 45)
[0286] [0286] Alternatively, the PCR product is subjected to the CEL | Enzyme Mutation Detection Assay, according to the instructions in the SURVEYOR Mutation Detection Kit (Transgenomics, USA). This assay is used to assess the effectiveness of the IPK1 DNA mutation through gene addressing by the programmed molecular complex.
[0287] [0287] Donor Sequence for Experiment 2A: GFP is fused to the IPK1 sequence and thus the expression of GFP can only happen by precise homologous (HR) recombination. The sequence of the entire donor sequence is similar to that described in SEQ ID No.: 46. The IPK1 homologous sequence required for recombination is nucleotides 1-621 and 1960-2610 of SEQ ID No.: 46, and the GFP cassette is encoded by nucleotides 622-1959.
[0288] [0288] Experiment 2B: IPK knockout and insertion of Bar, delivery to calluses
[0289] [0289] In this experiment, the genomic DSB in corn plants and the specific integration of the herbicide resistance bar gene that confers resistance to Bialophos (Phosphinothricin; Glufosinate-Ammonia; its analogues or commercial herbicides, such as, Basta, Bayer Crop Science) in the IPK1 gene that forms the knockout mutation and bar expression in the IPK1 locus, are tested. The programmed molecular complex forms the genomic DSB in the IPK1 sequence, initiating the integration of donor DNA into the IPK1 sequence through homologous recombination.
[0290] [0290] This example is performed on corn calluses that are transfected by DNA bombardment and then grown under Bialophos (Basta) selection.
[0291] [0291] Calluses bombarded with ssna modified SCNA, plasmid co-complicating protein fraction of the programmable molecular complex and donor DNA containing the resistance bar CDS expression cassette are grown in a regenerative medium containing 2.5mg / L from Bialaphos. Only calli, whose cells where the coding sequence of the bar gene are integrated with IPK1 through HR, are able to grow in these conditions, therefore, it is considered that plant material still proliferative after 1 month in this medium has its genome modified as wanted.
[0292] [0292] With this design, while the bar resistance cassette integrates the genome by HR to function properly, the Corynebacterium diphtheria toxin A cassette (DT-A) is an autonomous cassette that expresses DT-A under thermal shock conditions ( HS) (42 ° C). Thus, for further analysis, the calluses are divided into HS-induced calluses and uninduced calluses. Only calluses containing a perfect HR event do not express DT-A. Calluses that contain the plasmid randomly integrated, carrying both donor DNA and the DT-A cassette, express DT-A and consequently die.
[0293] [0293] In addition, the calluses are subjected to PCR analysis using the 1F and 1R primers shown in example 2A, followed by the digestion of the product, as above, with BspHl.
[0294] [0294] The Donor plasmid contains a bar resistance cassette, to be inserted in the IPK1 cleavage site, and a DT-A cassette that should not recombine in the IPK1 locus, as a marker of non-specific integration event: The resistance cassette bar resistance is flanked by sequences homologous in IPK1 (nts. 1-621 and 2338-2988 of SEQ ID: 47) necessary for HR, while cassette DT-A is located outside the site flanked by the homologous sequence. The bar cassette (No. 622-2337 of SEQ ID No. 47) contains a constitutive promoter 358 CaMv; CDS of the Streptomyces hygroscopicus bar gene for phosphinothricin acetyl transferase which confers resistance to ammonia glufosinate (nts. 1526-2078 of SEQ ID: 47); and the NOS terminator - downstream from the CDS bar.-The entire Donor 2B sequence is described in SEQ ID No.: 47.
[0295] [0295] On the same plasmid, a second cassette encoding diphtheria toxin A, DT-A, (from GenBank: AB535096.1) under the control of a heat shock inducible promoter (HS Promoter HSP18.2 GenBank: X17295.1) and terminated with a NOS terminator has the sequence as described in ID No.
[0296] [0296] The enzyme Phytoene Desaturase (PDS) is involved in the conversion of phytoene to zeta-carotene in the biosynthesis of carotenoid. Degradation of Arabidopsis phytene desaturates results in albino and dwarf phenotypes. This phenotype is explained by the biosynthesis of gibberellin, carotenoid and damaged chlorophyll. In this way, a mutation in this gene can be detected phenotypically.
[0297] [0297] In this example, a double chromosomal filament (DSB) break in the endogenous PDS gene is specifically induced in order to create a point mutation through a frame shift, and promoting the knock-out of the gene's function using the endogenous NHEJ pathway.
[0298] [0298] In this example, a chromosomal double-strand break (DSB) is specifically in the endogenous PDS gene in order to create an Insert of an mCherry Donor sequence in an endogenous PDS sequence to knock out PDS by assisted homologous recombination. using the programmable molecular complex.
[0299] [0299] For examples 3A-3B, a bioassay based on Arabidopsis protoplasts is used. In this bioassay, protoplasts are delivered with a plasmid that expresses the protein fraction of the molecular complex in vivo and are co-delivered with a pair of Specificity-Propelling Nucleic Acids (SCNA) ssDNA modified, in this example, by a Fluorescein (6-carboxy) terminal -Fluorescein, 6-FAM), each SCNA showing this modification at the 3'-terminal or at the 5 "-terminal (/ 86-FAM / and / 56-FAM /, respectively). A second modification to protect exonuclease, such as , phosphorothioate, is added at the opposite end, as it is possible to connect the internal phosphorothioate bonds for the protection of the endonu-
[0300] [0300] In this example, the protein fraction, encoded in a plasmid, contains an amino acid sequence adapted from a Fokl nuclease domain as a functional domain; an amino acid sequence adapted from an anti-Fluorescein (scFv) single-stranded variable fragment immunoglobulin (Protein Database access codes 1X9Q, 1FLR H), as Binding Domain; an SV40NLS (PKKKRKV: SEQ ID No.: 3) as a nuclear localization domain and a -15A interdomain connector (GGSGG: SEQ ID No.: 7).
[0301] [0301] Thus, the protein fraction of the molecular complex described in this example has the amino acid sequence as described in SEQ ID No.: 49 and is encoded by the nucleotide sequence described in SEQ ID No.: 50.
[0302] [0302] The specificity-promoting nucleic acid (SCNA) of this example is modified by the addition of a Fluorescein-ScFv / 6-FAM, 6-carboxyfluorescein - Fluorescein dT that includes a C6 / inker at one end of each SCNA.
[0303] [0303] The design of the SCNA essentially follows that described in Example 1. The length of the complementary base-pairing oligonucleotide SCNA preferably has at least 18 bases. The SCNA can also contain a small number (for example, 1-6, in one example, 6, in another example, 2) of unaddressed base pairing nucleotides (N's) of any sequence composition that serves as a spacer. between the modifier of the terminal 6-FAM and the complementary target nucleotides.
[0304] [0304] The target sequence is: ETCCTGCTAAGCCTTTGAAAG (SEQ ID: 51), Located in Exon 2 of the Arabidopsis PDS sequence (GIl: 5280985, gene dI3145c, protein id = "CAB10200.1).
[0305] [0305] SCNAs can be addressed to any strand, so for the target shown, there are 4 SCNA pairing options: SCNAs Direction (S):
[0306] [0306] PDS-SL1-846: GCATCCTTCCGTAGTGCTCCTCNNNNNN / 36-FAM / (nucleic acids are designated here only as SEQ ID NO: 52)
[0307] [0307] PDS-SR1-868: / 56-FAM / NNNNNNTTGTAATTGCTGGTGCTGGTAT (nucleic acids are designated here only as SEQ ID: 53) Anti-sense SCNAs:
[0308] [0308] PDS-ASL1-846: / 56-FAM / NNNNNNGAGGAGCACTACGGAAGGATGC (nucleic acids are designated here only as SEQ ID NO: 54)
[0309] [0309] PDS-ASR1-868: ATACCAGCACCAGCAATTACAANNNNNN / 36-FAM / (nucleic acids are designated here only as SEQ ID: 217) Mixed-strand SCNAs:
[0310] [0310] PDS-SL1-846: GCATCCTTCCGTAGTGCTCCTCNNNNNN / 36-FAM / (nucleic acids are designated here only as SEQ ID NO: 52)
[0311] [0311] PDS-ASR1-868: ATACCAGCACCAGCAATTACAANNNNNN / 36-FAM / (nucleic acids are designated here only as SEQ ID NO: 217)
[0312] [0312] PDS-SR1-868: / 56-FAM / NNNNNNTTGTAATTGCTGGTGCTGGTAT (nucleic acids are designated here only as SEQ ID NO: 53)
[0313] [0313] PDS-ASL 1-846: / 56-FAM / NNNNNNGAGGAGCACTACGGAAGGATGC (nucleic acids are designated here only as SEQ ID NO: 54)
[0314] [0314] A second group of paired combinations of "R" and "L" SCNAs,
[0315] [0315] PDS-SL2-845: TGCATCCTTOCCGTAGTGCTCCTNN / 36-FAM / (nucleic acids are designated here only as SEQ ID No.: 55)
[0316] [0316] PDS-SR2-870: / 5S6-FAMNNGTAATTGCTGGTGCTGGTATGT (nucleic acids are designated here only as SEQ ID NO: 56)
[0317] [0317] PDS-ASL2-845: / 56-FAM / NNAGGAGCACTACGGAAGGATGCA (nucleic acids are designated here only as SEQ ID No.: 57)
[0318] [0318] PDS-ASR2-870: ACATACCAGCACCAGCAATTACNN / 36-FAM / (nucleic acids are designated here only as SEQ ID No.: 58)
[0319] [0319] PDS-SL2-845: TGCATCCTTCCGTAGTGCTCCTNN / 36-FAM / (nucleic acids are designated here only as SEQ ID No.: 55)
[0320] [0320] PDS-ASR2-870: ACATACCAGCACCAGCAATTACNN / 36-FAM / (nucleic acids are designated here only as SEQ ID: 58)
[0321] [0321] PDS-SR2-870: / 56-FAMNNGTAATTGCTGGTGCTGGTATGT (nucleic acids are designated here only as SEQ ID NO: 56)
[0322] [0322] PDS-ASL2-845: / S6-FAM / NNAGGAGCACTACGGAAGGATGCA (nucleic acids are designated here only as SEQ ID No.: 57)
[0323] [0323] / 56-FAM / symbolizes a 5 'modification in the SCD ssDNA comprising 6-FAM (6-carboxy-Fluorescein). / 386-FAM / symbolizes a 3 'modification in the SCD ssD-NA comprising 6-FAM (6-carboxy-Fluorescein). N symbolizes any nucleotide.
[0324] [0324] Donor Sequence is DONOR PD-MCHERRY-S which has the sequence described in SEQ ID NO: 59 (ORF encoding mCherry is in nucleotides 662-1372 of SEQ ID NO: 59).
[0325] [0325] Bioassay configuration: The preparation of the Arabidopsis protoplasty is based on (Wu et. Al., 2009) and is similar to example 1 with differences in the transfection stage: Transfection:
[0326] [0326] In experiment 3A, the protoplast group DNA is analyzed by PCR and analysis of the restriction fragment of the PCR product.
[0327] [0327] PCR is conducted with three primers:
[0328] [0328] PCR Primer2F: TEGTTGTGTTTGGGAATGTTTCOT (SEQ ID NO: 60); and
[0329] [0329] BY Initiator2R: TATOCAAAAGATATCTTCCAGTAAAC (SEQ ID: 61)
[0330] [0330] The elimination of cleavage with the restriction enzyme Ddel in at least a portion of the amplified DNA indicates at least some success in gene addressing and targeted mutation of the genomic model.
[0331] [0331] In experiment 3B a Donor DNA encoding mCherry is fused in the frame to the endogenous PDS gene. The MRNA produced with this gene encodes a degraded PDS fused to a total mCherry immediately followed by a STOP codon ("PD-mCherry"). The protoplasts suspended in W5 solution are evaluated for mCherry activity 3 days after transfection using an automated flow cytometer (FACS) machine. PDS-modified protoplasts are detected through FACS analysis, where an insertion of the mCherry donor is detected by mCherry fluorescence using an excitation wavelength of 561 nm and emission detection at 590-630nm. Threshold and compensation factors are defined to exclude all false positives.
[0332] [0332] Additional characterization in both experiments is obtained by regenerating the protoplasts in the appropriate media and examining their posterior phenotypic character, when white plants or calluses indicate the success of gene addressing.
[0333] [0333] Substitution of the ALS gene in smoke and production of herbicide-resistant plants: Acetolactate synthase (ALS) is an enzyme in the biosynthetic pathways of valine, leucine, and isoleucine in plants. Mutations in this gene result in resistance to several herbicides. For example, mutations in the SuRB gene in smoke have been shown to provide resistance to herbicides: S647T - imazaquin, P191A - chlorsulfuron, WB568L - chlorsulfuron and imazaquin
[0334] [0334] In this example, ALS do Fumo is addressed in order to replace the wild type gene with a mutant herbicide-tolerant version through assisted substitution of the gene mediated by homologous recombination.
[0335] [0335] The expression and assembly of the molecular complex programmed in the tobacco plants are carried out in two stages. The delivery of the protein fraction is carried out by infecting a Tobacco Plant with a viral expression vector of the Fume Rattle Virus (TRV) protein, for example, a modified vector of pTRV2 (Vainstein et. Al., 2011) for delivery and expression of the programmable protein fraction in the plant.
[0336] [0336] SCNA delivery to plants that express the protein fraction is performed by infecting the plants with Agrobacterium carrying a T-DNA that encodes both a pair of RNA-SCNAs and a Donor sequence.
[0337] [0337] The RNA-SCNAs in this example bind to the protein fraction binding domain of the molecular complex using the bacteriophage Phi21l boxB 20-mer RNA binding sequence (SEQ No. 62: 5- UUCACCUCUAACCGGGUGAG- 3 ') as “SCNA nucleotide motif” schematically exemplified in Figure 1B.
[0338] [0338] The binding domain in this example is derived from the bacteriophage N protein Phi21 of the RNA binding protein (RBP) (SEQ ID NO: 63: GTAKSRYKARRAELIAER-C '). In the example shown here, the clamp is not on the target, but on the SCNA, and the action of the binding protein is therefore not restricted to a specific recognition site on the target RNA itself, but can be used to address any sequence, including DNA, depending exclusively on the target base pairing sequence of the variable SCNA adjacent to the invariant RBP binding clamp.
[0339] [0339] The target nucleic acid (gene) in this example is SuRB (GenBank accession Gl: 19778) and the desired amino acid mutation is P191A - conferring resistance to chlorsulfuron. Like this:
[0340] [0340] Original sequence unchanged: GGTCAAGTGCCACGTAGGATG (SEQ ID: 64)
[0341] [0341] Induced Mutation: GGTCAAGTGGCGCGCAGGATG (SEQ ID: 65) The sequence of the components of the protein fraction: Components:
[0342] [0342] Two options for assembling the protein are tested in this example:
[0343] [0343] Spatial measurements obtained from three-dimensional computerized models for the NP Phi21 C 'version in conjunction with the BoxB RNA clamp system and the GGSGGESK interdomain linker (SEQ ID: 74), as shown in this example, showed that the ideal distance expected between SCNAs, in the presence of a single "N" in the SCNA, corresponds to about 26-30 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and right of the 13th-17th nucleotide, counting begins after the nucleotide hybridizes to the SCNA in both
[0344] [0344] AAMAAAAAAAN DDD XXKKKXKKNNDIDIIVVVVVCOCCOCOCOCOCOCCC,
[0345] [0345] where Y + X represents the number of nucleotides between the SCNA base pairing sites, then the projected SCNAs pair with areas A and C and the cleavage resulting in the DSB is at or adjacent to area X. SCNAs they can be complementary to any of the felt and antisense filaments, however, they are chosen to pair preferentially with the sense (not transcribed) sequence. Both SCNAs can pair with the same strand, since the position of the protein fraction is located at the "near end" of the SCNA as defined by the 5 'or 3' modification of the primer that is at the "near end".
[0346] [0346] SCNAs pair with the flanking sequences of the target site to be cleaved in any of the strands, thus, for the target shown, using a target gap of 28bp: there are 4 SCNA pairing options: Sense pair of SCNA (S):
[0347] [0347] SURB P191 SR1 586:
[0348] [0348] UUCACCUCUAACCGGGUGAGNGGUACUGAUGCUUUUCAGGAAA (SEQ ID NO: 70)
[0349] [0349] SuURB. P191 SL1 557:
[0350] [0350] AUAGCGUCCCCAUUGUUGCUAUNUUCACCUCUAACCGGGUGAG (SEQ ID: 71) Pair of antisense SCNA (AS):
[0351] [0351] SuRB P191 ASR1 586:
[0352] [0352] UUUCCUGAAAAGCAUCAGUACCNUUCACCUCUAACCGGGUGAG (ID No.
[0353] [0353] SuRB P191 ASL1 557:
[0354] [0354] UUCACCUCUAACCGGGUGAGNAUAGCAACAAUGGGGACGCUAU (SEQ ID NO: 73)
[0355] [0355] And all combinations of sense and antisense pairs always choosing a SCNA Right (R) and Left (L):
[0356] [0356] The second option for the assembly of the protein tested in this example, assembled with Protein N Phi21 in the C 'of the protein and SV40NLS in the N' of the protein fraction. In this construct, an interdomain connector of the sequence: GGSGGESK (SEQ ID: 74) is used:
[0357] [0357] The assembled protein fraction based on NP Phi21 Mounted from this example has the amino acid sequence as described in SEQ ID No.: 75 and is encoded by the nucleic acid sequence as described in SEQ ID No.: 76.
[0358] [0358] The results of spatial measurements obtained with three-dimensional computerized models for the NP Phi21 C 'version together with the BoxB RNA clamp system and the GGSGGESK / interdomain linker (SEQ ID: 74), as used in this example, demonstrated that the ideal distance expected between SCNAs, in the presence of 1 N in the SCNA, corresponds to about 22-24 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and right of the 11th, 12th or 13th nucleotide, counting from the last nucleotide that hybridizes to the SCNA on both sides, taking into account the 5 'overhang (overhang) of base 4 created by dsDNA cleavage by the dimerized construct. This criterion suggests that, if the addressed sequence is, for this 23 nucleotide example: areas A and C and the cleavage resulting in the DSB is at or adjacent to area X. SCNAs can be complementary to any of the sense and antisense filaments, however, they are chosen to pair preferentially with the sense (not transcribed) sequence . Both SCNAs can pair with the same filament, since the position of the protein fraction is located at the "near end" of the SCNA as defined by the 5 'or 3' modification of the primer that is at the "near end". SCNA string options:
[0359] [0359] SCNAs pair with sequences flanking the target site to be cleaved in any strand, using a target gap of 31bp, resulting in 4 SCNA pairing options: SCNA Pair Direction (S):
[0360] [0360] SuRB P191 SR1-588: UUCACCUCUAACCGGGUGAGUACUGAUGCUUUUCAGGAAACU (SEQ ID: 77)
[0361] [0361] SuRB P191 SL1-556: GAUAGCGUCCCCAUUGUUGCUAUUCACCUCUAACCGGGUGAG (SEQ ID: 78) Pair of antisense SCNA:
[0362] [0362] SuURB P191 ASR1-588: AGUUUCCUGAAAAGCAUCAGUAUUCACCUCUAACCGGGUGAG (SEQ ID: 79)
[0363] [0363] SuRB P191 ASL1-556: UUCACCUCUAACCGGGUGAGUAGCAACAAUGGGGACGCUAUC (SEQ ID: 80) Combinations of sense and antisense pairs:
[0364] [0364] SuRB P191 SR1-588: UUCACCUCUAACCGGGUGAGUACUGAUGCUUUUCAGGAAACU (SEQ ID: 77)
[0365] [0365] SuRB P191 ASL1-556: UUCACCUCUAACCGGGUGAGUAGCAACAAUGGGGACGCUAUC (SEQ ID: 80)
[0366] [0366] SuRB P191 SL1-556: GAUAGCGUCCCCAUUGUUGCUAUUCACCUCUAACCGGGUGAG (SEQ ID NO:
[0367] [0367] SuURB P191 ASR1-588: AGUUUCCUGAAAAGCAUCAGUAUUCACCUCUAACCGGGUGAG (SEQ ID: 79)
[0368] [0368] A second group of paired combinations of "R" and "L" SCNAs, employing a 23bp target gap and a short SCNA linker (a single N) according to the prediction results:
[0369] [0369] Direction (S):
[0370] [0370] SURB P191 SR2-584: UUCACCUCUAACCGGGUGAGNUCGGUACUGAUGCUUUUCAGGA (SEQ ID: 81)
[0371] [0371] SURB P191 SL2-560: GCGUCCCCAUUGUUGCUAUAACNUUCACCUCUAACCGGGUGAG (SEQ ID: 82)
[0372] [0372] Anti-sense (AS): SuRB P191 ASR2-584:
[0373] [0373] UCCUGAAAAGCAUCAGUACCGANUUCACCUCUAACCGGGUGAG (SEQ ID NO: 83)
[0374] [0374] SuURB P191 ASL2-560: UUCACCUCUAACCGGGUGAGNGUUAUAGCAACAAUGGGGACGC (SEQ ID: 84)
[0375] [0375] or combinations of SCNAs "R" and "L" from the second group.
[0376] [0376] UUCACCUCUAACCGGGUGAG (SEQ ID: 62) is the sequence of the binding sequence of the boxi 20-mer RNA clamp of the bacteriophage Phi21, and acts as the connecting segment of the SCNA binding domain (marked schematically) as “SCNA nucleotide motif” in Figure 1B).
[0377] [0377] In this example, natural host plants, petunia, Nicotiana tabacum or N. Benthamiana are first inoculated with a vector based on pTRV- or pTRVdelta2b (Vainstein et. Al., 2011), which is designed to express, in this example, the programmable molecular construct under the control of a viral subgenomic promoter. About 5-21 days after infection, the leaves of the plant are collected and the plant sap, used here as an inoculum, is extracted by crushing the leaves in phosphate buffer (20OmM, pH 6.8), optionally supplemented with a non-ionic wetting agent, such as Silwet L-77 (about 0.015%). Clearing the solution by centrifugation and / or strainer is optionally followed by filtration at 0.22um. Filtration is necessary for injection into plants grown in tissue culture. Concomitantly, a portion of a leaf is analyzed for the stability of the viral construct by extracting the RNA, performing the reverse transcription of the RNA with a 3 'primer from the insertion site of the foreign gene, amplifying the CDNA by PCR using primers covering the strange gene insertion site and performing electrophoresis side by side in a similar way to the PCR amplified PTRV plasmid originally used for inoculation. The target tobacco plants, about 1 month old, are then infected by slight abrasion of the leaves with carborundum and rubbing the sap on the leaf surface. These plants can be grown in vitro or otherwise. The self-replication vector based on TRV carrying the programmable molecular complex infects the plant and promotes systemic dissemination to leaves, meristems and uninoculated tissues and organs. Although not yet programmed, said complex is inactive like the nuclease.
[0378] [0378] Once the self-replication vector based on
[0379] [0379] Analysis enabling the resolution of successful replacement events
[0380] [0380] In this example, specific methylation of DNA at a predetermined location is tested.
[0381] [0381] DNA methylation is catalyzed by DNA methyltransferases, which transfer a methyl group (-CH3) from S-adenosyl-L-methionine to the C-5 position of the cytosine residues. Three active DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B, have been identified in humans and mice. The methylation in these examples is from the DNA in the cytosine of a CpG sequence. These enzymes belong to a class of methyltransferases dependent on S-adenosylmethionine (SAM or AdoMet-MTase), class |; AdoMet-MTases are enzymes that use S-adenosyl-L-methionine (SAM or AdoMet) as a substrate for the transfer of methyl, creating the product S-adenosyl-L-homocysteine (AdoHcy).
[0382] [0382] DNMT3A
[0383] [0383] Both the DNMT1 and DNMT3 families of the methyltransferases contain the highly conserved C-5 methyltransferase motifs at their C-terminals, however they do not show sequence similarity in their N-terminal regions. DNMT3A also binds to deacetylases and is recruited by a sequence specific repressor to silence transcription. DNMT3A uses histone deacetylase HDAC1 using its ATRX homology domain. This domain of DNMT3A represents a repressive domain of independent transcription, whose silencing functions require HDAC activity. DNMT3A acts as the correct protein
[0384] [0384] In this example, a portion of the C 'of DNMT3A is used to construct a programmable molecular complex based on methyltransferase. PWWP domains that address DNMT3A to pericentric heterochromatin, zinc finger domains, ADD domains, ATRX region that causes its association with histone deacetylase HDAC1, and all N 'regulatory part of the protein are removed, maintaining the region comprising the AdoMet MTase region (www.uniprot.org Q9Y6K1). The C-terminal s DNMT3A and B contain the catalytic domain. In DNMT3A, the active site is C710 (number based on the translated access from GenBank AFO67972.2).
[0385] [0385] DNMT3A forms a heterotetramer complex DNMT3L: DNMT3A: DNMT3A: DNMT3L. DNMT3L is inactive like methylase, and DNMTS3A can dimerize and is active without DNMT3L. DNMTSA is functional in the homodimeric form. The complex shows specific contacts at the DNMT3A homodimer interface (dimer interface) and the dimerization brings two active enzyme sites separated by approximately one helix loop, in form B DNA. Thus, a dimer of the programmed molecular complex located in a specific SCNA / ocus can promote cytosine methylation at CpG sites at about 10-11 base pairs away. To further restrict the interactions of DNMT3A with DNMTL, the R729A mutation in the C 'terminal AdoMet MTase region is used in this example. The DNMT3A mutants that form dimers instead of tetramers in DNA are R771A, E733A, R729A, F732A, and Y735A.
[0386] [0386] In order to test the ability of the molecular complex in this example to perform specific targeted methylation on a predetermined DNA sequence,
[0387] [0387] The detection of transfected cells is performed by FACS analysis at a wavelength of 561nm excitation and emission detected by a 610/20 filter.
[0388] [0388] In this example, the protein, encoded in the delivered plasmid, contains an amino acid sequence adapted from the AdoMet MTase region containing the catalytic site of a methyltransferase based on human DNA (cytosine-5) -methyltransferase 38A (PDB access DNMTSA 2QRV is used to elucidate the three-dimensional structure). A mutation, R729 or R771 (based on GenBank translated numbering AFO67972.2) is added to eliminate tetramerization with the regulatory DNMTL without degrading the dimerization of DNMT3A or reducing Kcat. The amino acid sequence (translated according to GenBank AFO67972.2) from the methyltransferase region of this example is described in SEQ ID: 87 (AdoMet MTase region DNMT3A R729A)
[0389] [0389] An amino acid sequence adapted from the immunoglobulin of the single chain variable fragment of anti-Fluorescein (scFv) (access codes from Protein Database 1X9Q, 1FLR H) is used in this example as the Binding Domain; an SV40NLS (PKKKRKV: SEQ ID No.: 3) is used as a nuclear location domain and interdomain connectors, as well as a flexible interdomain connector (SEQ ID No. 14: GSLEGGSGG) are used in this example for its attachment.
[0390] [0390] The protein fraction has the amino acid sequence described in SEQ ID No.: 88, encoded by the nucleic acid sequence described as SEQ ID No.: 89:
[0391] [0391] The target sequence for the methylation assay is based on a mCherry coding cassette cloned into the MCS site of pSAT6-MCS (AY818383.1 Gl: 56553596) and includes the nucleotide sequence as described in SEQ ID No. :
[0392] [0392] SL898: TEGAGCTCAAGCTTCGAATTCTNNNNNN / 36-FAM / (nucleic acids are designated here only as SEQ ID NO: 91).
[0393] [0393] SR951: / 56-FAM / NNNNNNGATGGTGAGCAAGGGCGAGGAG (nucleic acids are designated here only as SEQ ID NO: 92).
[0394] [0394] The binding sites of the 3- and 5-6FAM (6 carboxy-Fluorescein) binding domain are marked by / 86-FAM / E / 56-FAM / respectively. Although one SCNA is sufficient for DNA methylation, it is possible to use more than one SCNA, correctly spaced to allow protein dimerization to increase specificity.
[0395] [0395] A double transfection strategy is used to allow expression of the protein fraction of the molecular complex before the introduction of SCNAs and Target DNA.
[0396] [0396] The preparation of the Arabidopsis protoplasty is based on Wu (Wu et. Al., 2009) and is similar to example 1 with differences in the transfection stage: Transfection:
[0397] [0397] Analysis of the CpG methylation condition of the target DNA applies two methods: A) The digested DNA from groups of protoplasts is analyzed by PCR amplification. Digestion is performed using restriction enzymes sensitive to methylation Smal (CCCGGG), Sall (GTCGAC) or Sacll (CCGCGG). The Smal, Sall, Sacll cluster is used as a CpG site for methylase. Underlined CpG dinucleotides. Methylated DNA is not cleaved by these enzymes. In this way, the sequence of MCS that covers the cleavage sites of these enzymes is amplified and the product measured by Quantitative PCR, providing a measure of the efficiency of methylation versus samples without the components of the molecular complex or deliberately containing non-specific SCNAs, precariously amplified due to complete cleavage resulting from non-methylation.
[0398] [0398] C-C chemokine receptor type 5 (CCR5, GenBank Access No. NT 022517.18) is a chemokine receptor expressed and displayed on the surface of T cells, macrophages, dendritic cells and microgliocytes. A mutation of this gene - CCR5-A32, which consists of a 32-base deletion, results in a frame shift mutation that introduces 31 new amino acids to the C ”-terminal of the truncated protein, and confers resistance to smallpox and some types of Viruses Human Disability (HIV). This allele is found in about 10% of Europeans, being rare in other groups.
[0399] [0399] In the example below, CCR5 or portions of this gene are excluded from hematopoietic stem cells (HSC) extracted from HIV-infected patients who do not have the A32 allele.
[0400] [0400] The protein fraction is formed by a functional Domain based on the nucleus (modified Fokl nuclease domain, as above) and a binding binding domain of the RNA motif (derived from the minimal TIV BIV peptide domain of the BIV TAT protein SGEPRPRGTRGKGRRIRR (SEQ ID NO: 93), where the protein fraction binding domain binds to the particular RNA sequence UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCU (SEQ ID: 94) which is the 1 BIV TAR loop. The nucleic coding for the protein fraction is carried out concurrently with the delivery of the specificity-promoting nucleic acid (SCNA) by the adenoviral vector, for its transient expression.The adenovirus is not integrated into the host genome.
[0401] [0401] After introduction and expression in target cells (HSC), molecular complexes autonomously assemble in the target gene CCR5, allowing fractions of the protein to dimerize and cleave the CCR5 sequence, to cause deletion of the portions of this gene as desired. Following this genetic modification, the genetically modified HSCs thus created, or their descendants, are re-transplanted autologously to the patient. The cells that have been modified are enriched by selection by removing the cells that exhibit CCRB5 before grafting. T cells and macrophages mutated by CCR5 that develop from these HSCs are resistant to HIV infection. Most of the adenoviruses and the molecular complex components were cleared from the HSCs before grafting, having completed their function.
[0402] [0402] Functional prevention of CCR5 display can be achieved through this system in a number of different ways, using different types and locations of SCNA, as detailed below:
[0403] [0403] In the A32 allele, 32 3 'nucleotides of COR5 CDS are absent, resulting in a frame shift deletion. The deleted string is: TTCCATACAGTCAGTATCAATTCTGGAAGAA (SEQ ID: 95). To exclude this sequence from cells expressing CCR5, SCNAs derived from the sequences below (shown without binding modification of the binding domain) are used:
[0404] [0404] ATCAATTCTGGAAGAATTTCCA (SEQ ID NO: 96);
[0405] [0405] TC “ATTACACCTGCAGCTCTCAT (SEQ ID NO: 97).
[0406] [0406] In this example, when the binding modification of the binding domain in a transcribed SCNA uses BIV TAR, the complete sequences of the SCNA sequences are:
[0407] [0407] SCNA 1 distance option, using a 16bp gap and none / "N" linker
[0408] [0408] CCR5 D32 SR 3321:
[0409] [0409] CCR5 D32 SL 3304:
[0410] [0410] SCNA 2 distance option, employing a 27bp target gap and 2 "N" linker nucleotides: COR5 D32 SR 3319:
[0411] [0411] UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCUNNGUAUCAAUUCUGGA AGAAUUUC (SEQ ID: 100)
[0412] [0412] CCR5 D32 SL 3291:
[0413] [0413] CAAMAAGAAGGUCUUCAUUACACNNUUCAGCUCGUGUAGCUCAUUVAG CUCCGAGCU (SEQ ID: 101)
[0414] [0414] These SCNAs are designed to allow modification / cleavage in the target sequence TITOCATACAGTCAGTATCAATTCTGGAAGAA (SEQ ID: 102). The cleavage and formation of DSB mediated by these pairs alone, in some cases, through endogenous mechanisms, can cause a mutation that would lead to a shift in the reading frame. In order to produce broader deletions in the CCNA5 CCR5 gene pairs, addressing at least two targets in CCRS5 are used:
[0415] [0415] Deletion of substantially the entire CCR5 coding sequence is induced using CCR5-ATG region binding SCNAs and CCR5 STOP codon region binding SCNAs, concurrently.
[0416] [0416] Area addressed between SCNAs (ATG underlined):
[0417] [0417] CAGGGTGGAACAAGATGGATTATCAAGTGTOC (SEQ ID: 103).
[0418] [0418] SCNA 1 distance option using a target gap of 31bp and no SCNA internal “N” linker:
[0419] [0419] CCR5 SR 2779:
[0420] [0420] CCR5 SL, 2747: AAGATCACTTTTTATTTATGCAUUCAGCUCGUGUAGCUCAUUVAGCUCCGAGCU. (SEQ ID No.: 105).
[0421] [0421] SCNA 2 distance option, based on computational results, using a target gap of 27bp and 2 nucleotides of the / inker “N” ":
[0422] [0422] CCR5 SR 2777:
[0423] [0423] UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCUNNUCAAGUCCAAUCUA UGACAUCA (SEQ ID: 106
[0424] [0424] CCR5 SL 2749:
[0425] [0425] GAUCACUUUUUAUUUVAUGCACANNUUCAGCUCGUGUAGCUCAUVAGC UCCGAGCU (SEQ ID: 107)
[0426] [0426] STOP SCNAs:
[0427] [0427] Area addressed between SCNAs (underlined STOP codon): ATATCTGTGGGCTTGTGACACGGACTCAAGT (SEQ ID: 108)
[0428] [0428] SCNA distance option 1 Using a target gap of 31bp and no SCNA internal “N” linker:
[0429] [0429] CCR5 SR 3884:
[0430] [0430] CCR5 SL 3802:
[0431] [0431] SOCNA 2 distance option, based on computational results, using a target gap of 27bp and 2 nucleotides of the linker “N”:
[0432] [0432] CCOR5 SR 3833:
[0433] [0433] UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCUNNUGGGCUGGUGACCÇ CAGUCAGAG (SEQ ID: 111)
[0434] [0434] CCR5 SL, 3805:
[0435] [0435] AUCCACUGGGGAGCAGGAAAUANNUUCAGCUCGUGUAGCUCAUUVAGC UCCGAGCU (SEQ ID: 112)
[0436] [0436] The protein fraction of the molecular complex is expressed via a nucleotide sequence carried in an Adenovirus-based expression system, such as Adeno-XTYM Adenoviral System 3 (Clontech Laboratories (CA, USA)) and observing it the manufacturer's instructions. Alternatively, the protein fraction is delivered by transfection of naked RNA.
[0437] [0437] Functional domain: derived from the Fokl nuclease subunit (as above).
[0438] [0438] Binding domain: minimum BPR TAT peptide domain SGPRPRGTRGKGRRIRR (SEQ ID: 93).
[0439] [0439] Cell location domain: SV40 nuclear location signal (NLS) domain (SV40NLS).
[0440] [0440] Fokl nuclease subunit: VKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHL GGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHI
[0441] [0441] SV40NLS: MPKKKRKV (SEQ ID NO: 67);
[0442] [0442] BIV TAT peptide: SGEPRPRGTRGKGRRIRR (SEQ ID NO: 93).
[0443] [0443] Interdomain connector: GSGGSGP (SEQ ID: 113)
[0444] [0444] The assembled BIV-based TAT protein fraction of this example has the amino acid sequence as described in SEQ ID No.: 114, which is encoded by the nucleic acid sequence as described in SEQ ID No.:
[0445] [0445] Spatial measurements obtained from computerized three-dimensional models for the BIV-TAT-TAR system with the GGSGGGP Interdomain / inker (SEQ ID: 116), as used in this example, demonstrated that the ideal distance expected between SCNAs, in the presence of 2 N's in the SCNA, corresponds to about 26-28 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and to the right of the 12th, 13th or 14th nucleotide, counting begins after the nucleotide hybridizes with SCNA on both sides, taking into account the 5 'overhang (overhang) of base 4 created by cleavage of dsDNA by the diffused construct. This criterion suggests that if, as in this example, the targeted sequence is 27 nucleotides:
[0446] [0446] AARAAAAAAANNVDYDVDVND "DDDXXKXXXXKVDDIWDDYYVCCCCOCCOCOCCC, where Y + X represents the number of nucleotides between the SCNA base pairing sites, then the projected SCNAs pair with the Ae Cea and cleavage areas resulting in the DSB area SCNAs can be complementary to any of the sense and antisense filaments, but are preferably chosen to pair with the sense (not transcribed) sequence. Both SCNAs can pair with the same filament, since the position of the fraction of protein is located at the "near end" of the SCNA as defined by modification 5 'or 3' of the primer which is at the "near end".
[0447] [0447] The detection and selection of cells that do not express / present wild-type vs. CCR5. cells expressing wild-type CCRS5 is performed by FACS analysis, using an anti-Human CCR5 mouse monoclonal antibody (R&D systems - Catalog No. FABSP1). EXAMPLE 7. Transcription Activator Addressed by Programmable Nucleic Acid Base Pairing
[0448] [0448] In this example, a protoplast system in monocotyledonous maize plants (Marrs & Urioste, 1995; Rhodes et. Al., 1988) is used as a bioassay. In this system, corn protoplasts are electroporated to introduce a plasmid for transient expression. These protoplasts can then be regenerated, if desired.
[0449] [0449] In this example, a protein fraction consisting of the Gal4 transcription activating domain, excluding the UAS binding domain, and a binding domain consisting of ScFv anti-fluorescein, together with a Fluorescein-modified SCNA, is used to activate the expression of a reporter gene. In this example, used here, the DNA binding domain of Gal4 is removed and replaced with a protein fraction binding domain.
[0450] [0450] In the first example, two reporter plasmids are used that can express GFP (option 1) or B-glucoronidase (GUS, option 2), only if a transcription activator is linked to a sequence upstream of a TATA box. In this example, this sequence is a 6X-UAS, known to be activated by the Gal4 protein.
[0451] [0451] In the second example, the UAS sequences are removed from the target nucleic acid and the SCNA binds to minus 62 (62nt downstream of the TATA box), achieving essentially the same result, but without any natural promoter. In the corn protoplasty bioassay system, the protein fraction shown below and the SCNA can be cotransfected using electroporation.
[0452] [0452] The amino acid sequence of the protein fraction: comprising a Gal4 activation domain addressed by nuclear N 'fused through an interdomain connector to a ScFv antifluorescein is here designated as SEQ ID No. 132 and is encoded by the sequence of nucleotide as described in SEQ ID No.: 157.
[0453] [0453] The first example uses a target plasmid with 6 UAS repetitions:
[0454] [0454] The target plasmid contains, in the following order (5 '-> 3'), a 6UAS promoter region followed by a TATA box and is designated here in SEQ ID No.: 180: GGACTGTAGAGGTTCCGGGTGACAGCCCTCCGACGGGTEGACAGCCCTCCGAC GGGTGACAGCCCTCCGAATTOTAGAGGATCCGGGTEACAGCCCTCCGACEGGT
[0455] [0455] AGGAAGTTCATTTCATTTGGRGAGGACACGCTGAACC (SEQ ID NO: 192); Option 1: A GFP coding sequence described in SEQ ID No.: 193. Option 2: A B-glucoronidase (GUS) coding sequence, described in SEQ ID No.: 194.
[0456] [0456] a Terminator-35 sequence:
[0457] [0457] GTCCGCAAAAATCACCAGTCTCTCTCTACAAATCTATOTCOTCOTCTATTITT TCTCCAGAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCTTATAGG
[0458] [0458] Two different SCNA orientations are provided in separate experiments to choose the most effective one: SOCNA for UAS sequence binding
[0459] [0459] Direction: CGGGTGACAGCCCTCCGANNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 196)
[0460] [0460] Anti-sense: / 5-8FAM / NNNNNNTCGGAGGGCTGTCACCCG (nucleic acids are only described here in SEQ ID No.: 197)
[0461] [0461] The end modification of SCNAs is 6-carboxy fluorescein (6FAM). Modification 5 'or 3' shown as / 5-6FAM / or / 3-6FAM / respectively. It does not represent any nucleotide.
[0462] [0462] The second example uses a target plasmid without a promoter to control the expression of the reported gene:
[0463] [0463] The target plasmid contains, in the following order, a sequence of the plasmid skeleton followed by a TATA box:
[0464] [0464] TOTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGA
[0465] [0465] A spacer sequence SEQ ID: 199): AGGAAGTTCATTTCATTTGGRGAGGACACGCTGAACC; Option 1: GFP ORF, as described in SEQ ID No.: 200.
[0466] [0466] Two different SCNA orientations are used:
[0467] [0467] SCNA: options (62 minus):
[0468] [0468] GCCAGGGTTTTCCCAGTCACGANNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 203)
[0469] [0469] / 50-85FAM / NNNNNNTCGTGACTGGGAAAACCCTGGC (nucleic acids are only described here in SEQ ID: 204)
[0470] [0470] Corn protoplasts are tested for GFP expression (option 1) using microscopic methods or flow cytometry. GFP positive cells indicate the functioning of the programmed complex. The percentage of cells positive to GFP allows to calculate the relative efficiencies between the experiments conducted to perfect different parameters of the system. The absence of GFP in cells lacking the appropriate components of the complex (for example, using non-specific control SCNAs) allows measuring the limits of specificity.
[0471] [0471] Corn protoplasts are tested for GUS expression (option 2) by staining the cells with X-Gluc in 0.45M mannitol and incubating overnight at 37ºC, and detected with a microscope. GUS positive cells (colored blue) indicate the functioning of the programmed complex. The percentage of GUS positive cells allows us to calculate the relative efficiencies between the experiments conducted to improve different system parameters. The absence of GUS in cells lacking the appropriate components of the complex (for example, using nonspecific control SCNAs) allows us to measure the limits of specificity.
[0472] [0472] In eukaryotes, organelles, such as mitochondria and plastids, contain their own genomes. Furthermore, in plants, organelles can also contain subgenomic circular DNAs. The modification of mitochondrial DNA can have implications for the treatment of human diseases and for use in agriculture, among others. The challenges of these modifications include, among other technical obstacles, the delivery and activation of a specific sequence system, reasonably efficient, necessary for the editing of the gene in the organelle.
[0473] [0473] PCF in Petunia
[0474] [0474] Cytoplasmic male sterility (CMS) is a valuable plant trait widely used by seed trading firms as a method to protect their seed strains. Thus, it is advantageous to repair the CMS in existing strains or to create CMS in new strains. Cytoplasmic male sterility can stem from failure in plants to produce functional male anthers, pollen, or gametes as a result of specific nuclear and mythondrial interactions. In the examples shown here, the trace of cytoplasmic male sterility characterized by petunia is used, which is caused by a conjugation of deletion and insertion in the atp9 gene in the mitochondrial DNA encoding the
[0475] [0475] The protein fraction of the programmable molecular complex in this example is designed to host a mitochondrial location signal in order to guarantee the location of the programmed molecular complex within the mitochondria. Other methods for transferring nucleic acids to the mitochondria include the use of liposomes or electroporation. The plant mitochondria, and specifically in a soybean plant, which includes Petunia, actively imports DNA through the permeability transition pore complex. The process is restricted to double-stranded DNA, but has no obvious sequence specificity. Donor sequences can be delivered, for example, as purified linear PCR fragments, linearized plasmids, or circular plasmids, depending on the delivery method. Expression from plasmids, electroporated in isolated wheat mitochondria, for example, is quite efficient when using a promoter compatible with the mitochondria, such as the 882 bp of the mitochondrial promoter cox Il of T. timopheevi containing the initiation region described by (Hanic-Joyce and Gray, 1991).
[0476] [0476] The selection of cells containing a substitution or insertion event can be obtained by a chloramphenicol resistance operon encoded in the Donor DNA.
[0477] [0477] In the examples (8A-8C) below, the protein fraction comprises:
[0478] [0478] A binding domain derived from the BIV TAT peptide comprising the amino acid sequence SEPRPRGTRGKGRRIRR (SEQ ID NO: 93);
[0479] [0479] A functional domain derived from the Fokl nuclease comprising the amino acid sequence VKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHL GGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHI NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGG
[0480] [0480] A cell localization domain derived from Arabidopsis lipoic acid synthase and comprising the amino acid sequence MHSRSALLYRFLRPASRCFSSSS (SEQ ID: 6) which is a mitochondrial localization signal (MLS).
[0481] [0481] Interdomain connector: GSGGSGP (SEQ ID: 113)
[0482] [0482] The assembled BIV TAT-based programmable protein fraction of this example has the amino acid sequence described in SEQ ID: 205, which is encoded by the nucleotide sequence described in SEQ ID: 206.
[0483] [0483] The results of spatial measurements obtained with three-dimensional computerized models for the BIV-TAT-TAR system with the GGSGGGP Interdomain / linker (SEQ ID: 116), in this example, shows that the ideal distance expected between SCNAs, in presence of 2 N's in the SCNA, is about 26-28 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and right of the 12th, 13th or 14th nucleotide, counting begins after the nucleotide hybridizes with SCNA on both sides, taking into account the 5 'protrusion (ove - rhang) of base 4 created by cleavage of dsDNA by the dimerized construct. This criterion suggests that, if the addressed sequence is, for example, the 27 nucleotides below:
[0484] [0484] AARMAAAAAAANNNNDVNDNDDDDXXKKXKXKKNVDIDIIIVVVVOCOCCOCCOCOCCCCC, where Y + X represents the number of nucleotides between the SCNA-based pairing sites, then the projected SCNAs pair with the areas A and DS can be cleaved to the area resulting in the cleavage resulting in the cleavage. complementary to any of the felt and antisense filaments, however, are chosen to pair preferentially with the sense (not transcribed) sequence. Both SCNAs can pair with the same filament, since the position of the protein fraction is located at the "near end" of the SCNA as defined by the 5 'or 3' modification of the primer that is at the "near end".
[0485] [0485] The binding RNA sequence of the SCNA Binding Domain used in this example is derived from loop 1 BIV TAR comprising the nucleic acid sequence UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCU (SEQ ID: 117). In this way the SCNA can be delivered directly to the isolated mitochondria (by electroporation of the mitochondria in the presence of a DNA encoding SOCNA under a bacterial promoter) or delivered to the cytoplasm (by transient transcription mediated by Agrobacterium) and “dragged ”To the mitochondria by the fraction of protein to which it is attached and comprising MLS.
[0486] [0486] After the expression of the programmable molecular complex, the mitochondria is isolated and a donor DNA is transfected into the isolated mitochondria.
[0487] [0487] The following examples are executed, each one having 2 options for the SCNA distances:
[0488] [0488] The target nucleic acid sequences for these examples include:
[0489] [0489] “ATP9” ": subunit 9 of the ATP mitochondrial synthase of Petunia x hybrid X Petunia axillaris subsp. Parodii, GenBank accession number Y00609.1 Gl: 297475.
[0490] [0490] “pcf”: Cytoplasmic male sterility (CMS) in Petunia axillaris subsp. parodii, CMS-associated fusion protein (CMS-afp), NADH dehydrogenase subunit 3 (nad3), and ribosomal protein S12 genes (rps12), complete cds; mitochondrial, GenBank accession number M16770.1 Gl: 1256946.
[0491] [0491] SCNAs are designed to form a single DSB at the target site, which is repaired by the endogenous NHEJ repair pathway, creating frame shifts in part of the coding sequence.
[0492] [0492] ATP9 target site: GCAAAACAATTATTTGGTTATGCCATTTTGG (SEQ No. | D: 118).
[0493] [0493] SCNA 1 distance option, target gap of 31bp:
[0494] [0494] SCNAs flanking the ATP9 target site:
[0495] [0495] ATP9 ASL 705:
[0496] [0496] ATP9 ASR 737:
[0497] [0497] SCNA 2 distance option, employing a 27bp target gap:
[0498] [0498] ATP9 ASL 707:
[0499] [0499] UUCAGCUCGUGUAGCUCAUUAGCUCCGAGCUGCCAAUGAUGGAUUU CGCGCCA (SEQ ID: 121)
[0500] [0500] ATP9 ASR 735:
[0501] [0501] AGCUUCGGEUUAGAGCAAAGCCCUUCAGCUCGUGUAGCUCAUUVAGCUC CGAGCU (SEQ ID: 122)
[0502] [0502] Petunia leaves are inoculated using a conventional method of leaf infiltration known in the art, with Agrobacterium housing T-DNA derived from a binary vector plasmid encoding the protein fraction, and RNA-SCNAs (as schematically shows the Figure 8A). After transfection, the components of the programmed molecular complex are expressed in the cytoplasm,
[0503] [0503] For analysis, 48 hours after transfection, the DNA is purified from the plants and the ATP9 sequence is amplified by PCR using primers:
[0504] [0504] ATP9atgF: ATGTTAGAAGGTGCAAAATCAA (SEQ ID: 123)
[0505] [0505] ATP9p2R: CTAARCGGACTTGGAATACGAAT (SEQ ID: 124)
[0506] [0506] The PCR product is then subjected to the CEL Enzyme Mutation Detection Assay | (SURVEYOR Mutation Detection Kit (Transgenomics, USA)). This assay is used to assess the effectiveness of the mitochondrial DNA mutation through gene addressing with a programmed molecular complex.
[0507] [0507] In this example, ATP9 is addressed to form a pcf-like mutant by inserting a donor DNA containing the chloramphenicol selection marker in the / ATP9 shell.
[0508] [0508] Method: as in example 8A, petunia leaves are inoculated using a conventional method of leaf infiltration with Agrobacterium that houses the T-DNA derived from a binary vector plasmid that encodes the protein fraction of the programmed molecular complex , and SCNAs. After transfection, the components of the programmed Molecular Complex are expressed in the cytoplasm, if they come together autonomously, and are located in the mitochondria by the mitochondrial import system through the MLS displayed on the surface of the protein fraction. After about 12-72 hours, the infiltrated leaves are used for mitochondrial preparation. A plasmid vector or a linear PCR product comprising the Donor DNA of this example, is delivered by electroporation to the isolated mitochondria. The electroporated mitochondria are then transplanted into fresh protoplasts of Petunia by microinjection. The injected protoplasts are regenerated in the chloramphenic selection medium, allowing only the PCF-like mitochondria to survive in the cells.
[0509] [0509] The 88 DNA DONOR (atp9 modified to resemble pcf) is described in SEQ ID: 125: Results and analysis:
[0510] [0510] The programmed molecular complex cleaves the atp9 gene in its co-complicating sequence, downstream from the pcf homologous region. This results in homologous recombination (HR) between the pcf-like donor and the cleaved atp9 gene. A male sterile pcf genotype in the mitochondrial genome is then recreated. In addition, the donor contains a chloramphenicol resistance cassette that allows the selection of the chloramphenicol resistant mitochondria. The protoplasts injected for regeneration in the selection medium containing chloramphenicol contain the mitochondria addressed with modified DNA. The callus resulting from these protoplasts is capable of differentiating buds, and finally whole plants are formed resulting in regenerated plants containing only the addressed mitochondria. In this way, sterile male Petunia is obtained from the regeneration of callus plants containing mitochondria resistant to chloramphenicol.
[0511] [0511] In this example, the pcf mutant is addressed to form an active repaired ATP9 sequence using a donor DNA bearing Chloramphenicol resistance.
[0512] [0512] In this example, the Donor DNA is designed to be integrated by HR into the pcf location, creating a STOP codon to recreate an intact ATP9 protein destined for the superfluous amino acid sequence that causes pcf dysfunction. A chloramphenicol resistance case (AY230218.1 Gl: 30267504) in the Donor DNA is used
[0513] [0513] Method: A plasmid vector comprising the Donor DNA of this example, the SCNA shown in example 8C and the protein fraction of example 8º, are delivered by electroporation to the isolated mitochondria, in this example in a single plasmid with a design similar to the one shown schematically in Figure 9.
[0514] [0514] Similar to example 8B, electroporated mitochondria are transplanted into Petunia protoplasts by microinjection. The protoplasts are seeded in a chloramphenicol selection medium. The callus resulting from these protoplasts is capable of differentiating sprouts (Frearson et. Al., 1973), and finally whole plants are formed, resulting in regenerated plants containing only the addressed mitochondria. These petunia plants are evaluated for male fertility.
[0515] [0515] Target site in pcf: AGACTTACATCACGATGTCTTTTTCITTCOGTT (SEQ ID: 126)
[0516] [0516] SONAs flanking the target site:
[0517] [0517] SCNA 1 distance option, target gap of 31bp:
[0518] [0518] CMS ASL 704:
[0519] [0519] CMS ASR 736:
[0520] [0520] SCNA 2 distance option, based on computational results, employing a 27bp target gap:
[0521] [0521] CMS ASL 706
[0522] [0522] uucageu CGUGUAGCUCAUUVAGCUCCGageuCUGUUAUUUGUAUAC- CUAACAC (SEQ ID: 129)
[0523] [0523] CMS ASR 734
[0524] [0524] ACGAMAACCAAAAUCAGAAUAAUUCAGCUCGUGUAGCUCAUUAGCUC CGAGCU (SEQ ID: 130)
[0525] [0525] The DONOR 8C sequence follows the description in SEQ ID No.: 131.
[0526] [0526] The FAS receptor (FasR), also known as the antigen of apoptosis 1 (APO-1, APT, TNFRSF6, CD95), is a protein that in humans is encoded by the TNFRSFG6 gene located on chromosome 10 in humans ( access Gen- Bank NC 000010 REGION: 90750288..90775542 GPC 000000034 VERSION NC 000010.10 Gl: 224589801). The Fas receptor is a death receptor displayed on the surface of cells that causes programmed cell death (apoptosis) forming the death-inducing signaling complex (DISC) by binding the ligand. The trimer of the Fas ligand anchored in the membrane on the surface of an adjacent cell causes the Fas receptor to trimerize. The Fas or FasL ligand (CD95L) is a homotrimeric type II transmembrane protein. Soluble FasL is less active than its membrane-bound counterpart and does not induce trimerization of the receptor and the formation of DISC. After resulting in the aggregation of the death domain (DD), the receptor complex is internalized and initiates a cascade of events through caspases, eventually culminating in DNA degradation, membrane blebbing, and other apoptosis characteristics. . This event can also be simulated by the binding of an agonistic Fas antibody, used in this example.
[0527] [0527] Eight splice variants of FasR are known and are translated into the protein isoforms. The apoptosis-inducing Fas receptor is isoform 1, which is a type 1 transmembrane protein. The Fas protein has 319 amino
[0528] [0528] The protein fraction follows the description in Example 3.
[0529] [0529] Therefore, the protein fraction of the molecular complex described in this example has the amino acid sequence described in SEQ ID No.: 49.
[0530] [0530] The specificity-promoting nucleic acid (SCNA) of this example is modified by the addition of a Fluorescein-ScFv / 6-FAM, 6-carboxyfluorescein - Fluorescein dT that includes a C6 / linker at one end of each SCNA.
[0531] [0531] The length of the complementary target base pairing oligonucleotide SCNA is preferably at least 18 bases. The SCNA may further contain a small number (for example, 0-6, in this example, 6) of unaddressed base pairing nucleotides (N's) of any sequence composition that serves as a spacer between the 6-FAM modifier terminal and complementary target nucleotides.
[0532] [0532] The results of spatial measurements obtained with computerized three-dimensional models for the anti-Fluorescein-ScFv-6-FAM system with the GGSGG interdomain / linker (SEQ ID: 7), as used in this example, demonstrated that the expected ideal distance between SCNAs, in the presence of 2 N's in the SCNA, corresponds to about 23-26 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and right of the 11th, 12th or 13th nucleotide, counting from the last nucleotide that hybridizes with the SCNA on both sides,
[0533] [0533] AAMAAAAAAA DDD XXXXXXYYYYYYYCCCCCCOCCCCO, where Y + X represents the number of nucleotides between the SCNA based pairing sites, then the projected SCNAs pair with areas A and C and the cleavage resulting in the DSB is in or adjacent to the area X. SCNAs can be complementary to any of the sense and antisense filaments, however, they are chosen to pair preferentially with the sense (not transcribed) sequence. Both SCNAs can pair with the same filament, since the position of the protein fraction is located at the “near end” of the SCNA as defined by the 5 'or 3' modification of the primer that is at the “near end ”.
[0534] [0534] Examples of target sequences are: A) Exon 1 starts at 347, the target sequence is: GGGCATCTG GACCCTCCTACC (SEQ ID: 133)
[0535] [0535] SCNAs:
[0536] [0536] SCNA 1 distance option, 21bp SL351 target gap: A * GGATTGCTCAACAACCATGCTNNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 134)
[0537] [0537] SR373: / 56-FAM / NNNNNNTCTGGTGAGCCCTCTCCTGCC * C (nucleic acids are only described here in SEQ ID: 135)
[0538] [0538] SCNA 2 distance option, based on computational results, employing a 24bp target gap and a shorter SCNA / inker “N”:
[0539] [0539] SL349: G * 'GAGGATTGCTCAACAACCATGNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 136)
[0540] [0540] SR374: / 58-FAM / NNCTGGTGAGCCCTCTCCTGCCC * G (nucleic acids are only described here in SEQ ID: 137)
[0541] [0541] Exon 2 starts at 12499, the target sequence is: TACGTCTGTTGCTAGATTATC (SEQ ID: 138) B)
[0542] [0542] SCNAs:
[0543] [0543] SCNA 1 distance option, target gap with 21bp:
[0544] [0544] SL12503: A * TGCTTTTATTTTACAGGTTCTNNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID No.: 139)
[0545] [0545] SR12525: / 56-FAM / NNNNNNGTCCAAAAGTGTTAATGCCCA * A (nucleic acids are only described here in SEQ ID: 140)
[0546] [0546] SCNA 2 distance option, based on computational results, employing a 24bp target gap and a shorter SCNA / N "inker:
[0547] [0547] SL12501: TOATGCTTTTATTTTACAGGTTNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 141)
[0548] [0548] SR 12526: / 56-FAM / NNTCCAAAAGTGTTAATGCCCAA '* G (nucleic acids are only described here in SEQ ID: 142)
[0549] [0549] Exon 2 Target for restriction analysis: CAGTITGAGACTCAGAACTTGG (SEQ ID: 143) C)
[0550] [0550] SCNAS:
[0551] [0551] SCNA 1 distance option, 21bp target gap
[0552] [0552] SL12595: G * GAATTGAGGAAGACTGTTACTANNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 144)
[0553] [0553] SR12617: / 56-FAM / NNNNNNAAGGCCTGCATCATGATGGCCAATTCT * C (nucleic acids are only described here in SEQ ID: 145)
[0554] [0554] SOCNA 2 distance option, based on computational results,
[0555] [0555] SL12594: G "GAATTGAGGAAGACTGTTACTNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 146)
[0556] [0556] SR12619: / 56-FAMWNNGGCCTGCATCATGATGGCCAA * T (nucleic acids are only described here in SEQ ID: 147)
[0557] [0557] Initiators for analysis of example C:
[0558] [0558] FAS. E2F: CATGCTTTTATTTTACAG; (SEQ ID No: 148)
[0559] [0559] FAS E2R: CTGTGACTTTCACTGTAATC (SEQ ID: 149)
[0560] [0560] The PCR amplification of the target with these primers forms (in unmodified DNA) a 227bp PCR product digested with Ddel forming fragments of 127bp and 100bp. Digestion with Ddel is eliminated by precise addressing.
[0561] [0561] Exon 9 target: CAATTGTGAATTCACATAGAA (SEQ ID: 150) D)
[0562] [0562] SCNAs:
[0563] [0563] SCNA 1 distance option, target gap with 21bp
[0564] [0564] SL24524: G * GTGTCATATTATACAATATTTNNNNNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 151)
[0565] [0565] SR24546: / 56-FAM / NNNNNNAACATTAAATTATAATGTTTG * A (nucleic acids are only described here in SEQ ID: 152)
[0566] [0566] SOCNA 2 distance option, based on computational results, employing a 24bp target gap and a shorter SOCNA “N” inker:
[0567] [0567] SL24522: T'TGEGTGTCATATTATACAATATNN / 36-FAM / (nucleic acids are only described here in SEQ ID: 153)
[0568] [0568] SR24547: / 56-FAM / NNACATTAAATTATAATGTTTGA'C (nucleic acids are only described here in SEQ ID: 154)
[0569] [0569] Initiators for analysis of example D:
[0570] [0570] FAS. E9GF CTTTGTTTATAACTCTGAGAAG (SEQ ID No: 155)
[0571] [0571] FAS E9R TOCAAAATGCTTTTGATGCCTGA (nucleic acids are only described here in SEQ ID: 156)
[0572] [0572] The PCR amplification of the target with these primers forms (in unmodified DNA) a 240bp PCR product digested with EcoRI! forming fragments of 134bp and 106bp. Digestion with EcoR! is eliminated by precise addressing.
[0573] [0573] / 56-FAM / and / 386-FAM / symbolize a 5 'or 3' modification respectively in the SCNA ssDNA formed by 6-FAM (6-carboxy-Fluorescein). N symbolizes any nucleotide. Phosphorothioate bonds are symbolized by an asterisk (*).
[0574] [0574] Although each pair of SCNAs can cause a mutation to knock out the FAS receptor, deletion of an entire length of DNA resulting from addressing, more than one site in the gene can instantly disable FASR activity. Thus, for example, the use of SCNAs in the AC examples can result in mutations that eliminate FasR activity, while the use of any of these SCNAs in conjunction with the SCNA in example D leads to a greater genomic deletion that eliminates FasR activity.
[0575] [0575] Below is a bioassay to detect a specific mutation induced in human genomic DNA: HeLa and Jurkat cells are transfected with a plasmid that encodes the protein fraction of the programmable molecular complex together with the relevant ssDNA SCNAs. transfection (Mirus, USA) TransIT-HeLaMONSTER or TransIT-LT1 to formulate the plasmid DNA and TransIT-Oligo to formulate the SCNA ssDNA. Once incubated for the prescribed time, the two groups of mixtures of the formulated DNA transfection agent are supplied simultaneously to the cells, to address the chromosomal FasR. To determine the efficiency of gene addressing, cells are tested for their sensitivity to Fasl in a protocol modified from
[0576] [0576] This example refers to a bioassay suitable for testing and adjusting permutations in the basic design of the programmable molecular complex; test its application in different organisms or cells; test different delivery methods; and test the mutation, substitution, deletion and insertion editing functions.
[0577] [0577] Selectable bacterial marker genes are used to determine the efficiency of gene targeting when addressing plasmid DNA.
[0578] [0578] In these examples, a bioassay based on Arabidopsis protoplasts is used. In this bioassay, the protoplasts are delivered together with the reporter system and the molecular complex in a plasmid, co-delivered with SCNA ssDNA paired modified with a Digoxigenin (NHS Ester) (DIG) terminal, a SCNA that has this modification in the 3 "- terminal and the other in the 5 "-terminal. A second modification to protect exonuclease, such as phosphorothioate, can be added to the opposite end. Protein sequence and properties
[0579] [0579] The protein fraction follows the description in Example 1.
[0580] [0580] In this example, the modification at the nucleic acid end of the SCNA is a NHS-Ester-linked Digoxigenin (DIG), attached to the 5 'or 3' position of the oligonucleotide.
[0581] [0581] The amino acid sequence (one letter code) of the protein fraction of the molecular complex (NLS-Fokl-nuclease sequence With Digoxigenin ScFv is described in (SEQ ID: 12): SCNA properties and sequence
[0582] [0582] The length of the complementary target base pairing oligonucleotide SCNA is preferably at least 18 bases. The SCNA can also contain a small number (for example, 1-6, in one example, 6, in another example, 2) of unaddressed base pairing nucleotides ("N's") of any sequence composition that serves spacer between the DIG-NHS terminal modifier and the complementary target nucleotides.
[0583] [0583] The results of spatial measurements obtained with three-dimensional computerized models for the anti-DIG-ScFv-NHS-Ester-DIG system with the GSLEGGSGG interdomain / linker (SEQ ID: 14), as shown in this example, showed that the ideal distance expected between SCNAs is, in the presence of 2 N's in the SCNA, about 23-26 nucleotides. Cleavage is expected to occur about +2 nucleotides to the left and right of the 11th, 12th or 13th nucleotide, counting from the last nucleotide that hybridizes to the SCNA on both sides, taking into account the 5 'overhang (overhang) of base 4 created by dsDNA cleavage by the dimerized construct. This criterion suggests that, if the addressed sequence is, for this example of 24 nucleotides:
[0584] [0584] AAMAAAAAAA ND XXKKXXXXVYYYYYYYYCCCCCCCCCC, where Y + X represents the number of nucleotides between the SCNA based pairing sites, then the projected SCNAs pair with areas A and C and the cleavage resulting in the DSB is in or adjacent to the area X. SCNAs can be complementary to any of the sense and antisense filaments, however, they are chosen to pair preferentially with the sense (not transcribed) sequence. Both SCNAs can pair with the same filament, since the position of the protein fraction is located at the “near end” of the SCNA as defined by the 5 'or 3' modification of the primer that is at the “near end ”.
[0585] [0585] The target plasmid pTGD (represented schematically in Figure 15) comprises 4 main sections:
[0586] [0586] This plasmid multiplies in bacterial cells, for example, E. Coli cells. In this example, SCNAs, the target plasmid pTDG encoding the protein fraction of the programmable molecular complex and a donor DNA (in examples 10B, 10C) are delivered in Arabidopsis protoplasts. 48 hours after transfection, DNA is extracted from the transfected protoplasts (Kit A1120 Promega Corp.) and transformed into competent E. Coli bacterial cells (Kit L3002 Promega Corp.). The transfected bacteria are spread on LB medium containing kanamycin at a concentration of 100 micrograms / ml. Colonies are grown for about 16h at 37 ºC. The colonies are then copied to LB plates of Ampicillin (100 micrograms / ml) or Tetracycline (100 micrograms / ml) and grow for an additional 16h at 37 ºC. Analyze:
[0587] [0587] The colonies of each copy are counted. The number of kanamycin-resistant colonies suggests the total number of plasmids, which also represents the total target number. Colonies not resistant to Ampicillin are colonies that contain a plasmid successfully addressed, validating the editing functions of “Mutation” or “Deletion”. Colonies resistant to Tetracycline, however, not to Ampicillin, represent the integration of donor DNA into the target plasmid by NHEJ validating the “Substitution” editing function. Colonies resistant to Ampicillin and Tetracycline are colonies containing plasmids that were addressed, had the donor integrated into the Ampicillin target sequence, but did not replace it, validating the “Insert” editing function.
[0588] [0588] The plasmids are then subjected to PCR and sequence analysis to verify the results with the primers:
[0589] [0589] A961F: TAGGEGCGCTGGCAAGTGTAG (SEQ ID No.: 158)
[0590] [0590] A2161R: CATAACACCCCTTGTATTAC (SEQ ID NO: 159) Experiments: Example 10A - Mutation addressed in the AMPR cassette.
[0591] [0591] The detection assay is performed essentially as described above (“detection assay”) with the following additional details: plasmid pTGD is transfected together with SCNAs flanking target sequence 1 (ID No.
[0592] [0592] The detection assay is performed essentially as described above (“detection assay”) with the following additional details: the plasmid pTGD is transfected together with the SCNAs flanking target sequence 1 and a Tetracycline donor (Tet ) with linear dsDNA, produced as a PCR product, in Arabidopsis protoplasts. DNA is purified and transformed into competent E. Coli cells that are spread on KM LB medium. A copy is made on LB AMP and Tet plates. Colonies that lose resistance to AMP contain an addressed plasmid. Tet-resistant colonies represent plasmids containing specifically integrated donor DNA.
[0593] [0593] The detection assay is performed essentially as described above (“detection assay”) with the following additional details: the plasmid pTGD is transfected together with SCNAs addressed against target sequence 1 and SCNAs against target sequence 2 (SEQ ID: 170), together with the Tetracycline donor DNA (Tet) to the Arabidopsis protoplasts. The DNA is purified and transformed into competent E. Coli cells that are spread in KM LB medium. A copy is made on LB AMP plates and Tet LB plates. Colonies that lose resistance to AMP contain an addressed plasmid. Tet-resistant colonies represent specifically integrated donor DNA. Colonies sensitive to AMP are subjected to PCR analysis with A961F and A2161R primers.
[0594] [0594] Colonies containing a plasmid incorporating the Tet donor (ca. 1.9Kb) instead of the AMP target sequence (ca. 860bp) demonstrate gene replacement events
[0595] [0595] Colonies sensitive to AMP and Tet demonstrate deletion of the gene by NHEJ.
[0596] [0596] Tet and AMP resistant colonies contain a plasmid incorporating the TetR donor without the deletion of the Amp resistance cassette and demonstrate integration or “insertion” of the addressed donor. Delivery
[0597] [0597] Bioassay configuration: the preparation of the Arabidopsis protoplasty is based on Wu et. al. (2009), and is similar to example 1 with differences in the transfection stage: Transfection:
[0598] [0598] The protoplasts are then subjected to DNA extraction as described in the Detection Assay.
[0599] [0599] The addressed AMpR cassette follows the description in SEQ ID: 160.
[0600] [0600] SCNA pairs are chosen one left (L) and one right (R) regardless of the sense (S) or antisense (AS) filament: The choice of the combination of the SCNA pair is a parameter tested in experiment.
[0601] [0601] Target sequence T1 on the AMPR cassette: TATGAGTATTCAACATTTCCG (SEQ ID: 161) (initial ATG codon is underlined) Group 1 of AMP addressing SCNAs: Option 1 - using a target gap with 21bp:
[0602] [0602] DTGD 130 SL: A * ATAATATTGAAAAAGGAAGAGNNNNNN / 3DIGN / (nucleic acids are only described here in SEQ ID: 162)
[0603] [0603] DTGD 152 SR: / SDIGNANNNNNNTGTCGCCCTTATTCCCTTITITTT (nucleic acids are only described here in SEQ ID: 163)
[0604] [0604] vTGD 130 ASL: / SDIGN / NNNNNNCTCTTCCTTTTITTCAATATTAT * T (nucleic acids are only described here in SEQ ID: 164)
[0605] [0605] vTGD 152 ASR: AAMAAAGGGAATAAGGGCGACANNNNNN / 3DIGN / (nucleic acids are only described here in SEQ ID: 165) Option 2 - paired combinations, employing a 24bp target gap and a shorter SCNA linker according to the prediction results: AMP 129 SL: C * AATAATATTGAAAAAGGAAGANN / 3DIGN / (nucleic acids are only described here in SEQ ID: 166)
[0606] [0606] AMP 154 SR: / SDIGNANNTCGCCCTTATTCCCTTTTTTG * C (nucleic acids are only described here in SEQ ID: 167)
[0607] [0607] AMP 129 ASL: / SDIGN / NNTCTTCCTTTTTCAATATTATT * G (nucleic acids are only described here in SEQ ID: 168)
[0608] [0608] AMP 154 ASR: G'CAMAAAAGGGAATAAGGGCGANN / 3DIGN / (nucleic acids are only described here in SEQ ID: 169)
[0609] [0609] Target sequence T2 in the AMPR cassette: AGCATTGGTAACTGTCAGACC (SEQ ID: 170) Group 2 of AMP addressing SCNAs: Option 1 using a target gap with 21bp:
[0610] [0610] pvTGD 981 SL: G * AGATAGGTGCCTCACTGATTANNNNNN / 3DIGN / (nucleic acids are only described here in SEQ ID: 171)
[0611] [0611] DTGD 1003 SR: / SDIGNINNNNNNAAGTTTACTCATATATACTTT * A (nucleic acids are only described here in SEQ ID: 172)
[0612] [0612] DTGD 981 ASL: / SDIGNANNNNNNTAATCAGTGAGGCACCTATCT * C (nucleic acids are only described here in SEQ ID: 173)
[0613] [0613] DTGD 1003 ASR: T * AMAGTATATATGAGTAAACTTNNNNNN / 3DIGN / (nucleic acids are only described here in SEQ ID: 174) Option 2 paired combinations, employing a 24bp target gap and a shorter SCNA linker according to the prediction results:
[0614] [0614] AMP 980 SL: T "GAGATAGGTGCCTCACTGATTNN / SDIGN / (nucleic acids are only described here in SEQ ID: 175)
[0615] [0615] AMP 1005 SR: / SDIGN / NNGTTTACTCATATATACTTITAG * A (nucleic acids are only described here in SEQ ID: 176)
[0616] [0616] AMP 980 ASL: / SDIGN / NNAATCAGTGAGGCACCTATCTC * A (nucleic acids are only described here in SEQ ID: 177)
[0617] [0617] AMP 1005 ASR: T * CTAMAGTATATATGAGTAAACNN / 3DIGN / nucleic acids are only described here in SEQ ID: 178) Donor:
[0618] [0618] Donor sequence encoding the tetracycline resistance of the pSoup cloning vector, EUO48870.1 GlI: 155733614 accompanies the description in SEQ ID No.:
[0619] [0619] In this example, the programmable molecular complex is designed to operate with a single nucleic acid molecule incorporating the double target sequence that binds to the nucleic acid sequences, hereinafter referred to as a connected pair of Specificity-Enabling Nucleic Acid Sequences (SCNA sequences) as schematically illustrated in Figures 4A and 4B.
[0620] [0620] In this example, a degraded GFP target sequence is repaired by removing or mutating a STOP codon. The resulting cleavage of predetermined GFP-Target leads to point mutation that can restore GFP activity.
[0621] [0621] In these examples, a bioassay based on Arabidopsis protoplasts, in which the protoplasts are delivered together with the reporter system (target plasmid), plasmid that expresses the protein fraction, co-delivered with: For example, 12A (illustrated schematically in Figure 4A) - A nucleic acid encoding an RNA, RNA formed by two SCNA sequences modified, in this example, by the binding sequence of the boxi 20-mer RNA clamp of the bacteriophage Phi21 (SEQ ID: 62: -UUCACCUCUAACCGGGUGAG-3 ') and a “SCNA Connector”, an extension of non-targeting nucleotides of indefinite sequence or length. A SCNA with this modification at the 3-terminal and the other at the 5 "- terminal of the RNA molecule. The RNA-SCNAs, in this example, bind to the binding domain of the protein fraction of the two molecular complexes using the sequence of binding of the boxB 20-mer RNA clamp of the bacteriophage Phi21 (5 "- UUCACCUCUAACCGGGUGAG-3 '(SEQ ID: 62), or:
[0622] [0622] In Example 11B (illustrated schematically in Figure 4B), an SCD modified by ssDNA containing the sequence, in this example, modified at both the 5 'and 3' ends by the addition of the Digoxigenin (NHS Ester) (DIG) terminal molecules and a “SCNA Connector”, a non-targeting nucleus hybridization extension
[0623] [0623] The protein fraction in example 11A, contains an amino acid sequence derived from a Fokl nuclease domain as a functional domain, The binding domain derived from the N protein of the bacteriophage Phi21 of the RNA-binding protein (RBP) (No. SEQ ID: 683: N-GTAKSRYKARRAELIAER-C '), an SV40NLS (PKKKRKV: SEQ ID No.: 3) as a nuclear location domain and an interdomain connector (SEQ ID No.: 14: GSLEGGSGG).
[0624] [0624] The protein fraction in example 11B contains an amino acid sequence adapted from a Fokl nuclease domain as a functional domain; an amino acid sequence adapted from anti-DIG (scFv) single-chain variable fragment immunoglobulin (DIG-ScFv) similar to that described in (Huston et. al., 1988) as the Binding Domain; an SV40NLS (PKKKRKV: SEQ ID No.: 3) as a nuclear location domain and an interdomain connector (SEQ ID No.: 14: GSLEGGSGG).
[0625] [0625] The modifications of the nucleic acid end of the SCNA are NHS-Ester-linked Digoxigenin (DIG) and are attached at the 5 'and 3' positions of the oligonucleotide.
[0626] [0626] Protein = ”B of Phi21 Bacteriophage (SEQ ID: 63: GTAKSRYKARRAELIAER) at or near the N'-terminal as in the total length of the N protein, the RNA-binding is located at the N-terminal.
[0627] [0627] Nuclease Fokl !:
[0628] [0628] VKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYG YRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQ TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVE
[0629] [0629] SV40-NLS: (PKKKRKV: SEQ ID NO: 3)
[0630] [0630] Interdomain connectors: several polyamino acid linkers are tested for the optimal function of the programmed molecular complex.
[0631] [0631] Amino acid sequence of the protein fraction of the molecular complex: In this example, the N Phi21 protein (amino acid sequence as described in SEQ ID: 68) is assembled at the N'-terminal of the protein fraction of the molecular construct programmable and the nuclear location signal, SV40NLS, is located at the C 'terminal and the interdomain linker is GGSGG (SEQ ID: 7).
[0632] [0632] The SCNA length of the complementary target base pairing oligonucleotide can have any predetermined length. For example, the length can be at least 18 bases. The SCNA can also contain a small number (preferably 0-6, more preferably 1-2) of unaddressed base pairing nucleotides (N's) of any sequence composition that serves as a spacer between the A) terminal modifier boxB RNA clip Phi21 in example 11A or 11B) DIG-NHS terminal modifier in example 12B, and complementary nucleotides. In these examples, SCNAs are connected by a non-target base pairing sequence called “SCNA Connector” in Figure 14 or X (n) in the sequences in this example. X (n) means an indeterminate length of RNA nucleotides that connect the two regions that provide specificity to each other. For linear DNA, the ideal expected length (n) is about 35-73 nucleotides (nts), although longer (above 73 nts) and shorter (4-34 nts) SCNA connectors are apply it
[0633] [0633] SCNAs can be complementary to any of the felt and antisense filaments, however, they are chosen to pair preferentially with the felt (not transcribed) sequence although two options are shown here for each example. The two SCNA sequences can pair with the same strand, since the position of the protein fraction is located at the “near end” of the SCNA as defined by the 5 'or 3' modification of the primer that is at the “near end”.
[0634] [0634] "STOP GFP" Target containing the plasmid for the tests of Examples 11A and 11B contains the nucleic acid sequence as described in (SEQ ID No.: 181).
[0635] [0635] Dual sense or antisense hybridization SCNAs are constructed: Connected sense SCNAs:
[0636] [0636] GFP-921SR-X (n) -892SL BOXBPHI
[0637] [0637] UUCACCUCUAACCGGGUGAGNUCCAAGGGCGAGGAGCUGUUCA (SEQ ID NO: 207) -X (n) -ACCAUUUACGAACGAUAGCCAUNUUCACCUCUAACCGGG UGAG (designated as SEQ ID No.: 208).
[0638] [0638] GFP-921 ASR-X (n) -892ASL BOXBPHI
[0639] [0639] UUCACCUCUAACCGGGUGAGNAUGGCUAUCGUUCGUAAAUGGU (SEQ ID NO: 209) -X (n) -UGAACAGCUCCUCGC CCUUGGANUUCACCUCUAACCGGG UGAG (SEQ ID NO: 210)
[0640] [0640] The PHI boxB 20-mer 5-UUCACCUCUAACCGGGUGAG-3 'sequence (SEQ ID: 62) is underlined. Sequences that give specificity to the double SCNA are indicated in the schematic drawings of Figures 4A-B to SCNA1 and SCNA 2. N's means a short extension (0-6) of any nucleotide, X (n) means a non-targeting hybridization extension of indefinite sequence or length (SCNA Connector). Example 11B:
[0641] [0641] Dual sense or antisense hybridization SCNAs are constructed: Connected sense SCNAs:
[0642] [0642] GFP-919SR-X (n) -894SL-DIG
[0643] [0643] / S5DIAON / NNNTGTCCAAGGGCGAGGAGCTGTT (only nucleic acids are designated as SEQ ID No.: 211)
[0644] [0644] -X (n) - CATTTACGAACGATAGCCATGGNN / 3DigN / (only nucleic acids are designated as SEQ ID No.: 212) Connected antisense SCNAs:
[0645] [0645] GFP-919ASR-X (n) -894ASL-DIG
[0646] [0646] / S5DIAONNNNCCATGGCTATCGTTCGTAAATG (only nucleic acids are designated as SEQ ID No.: 213) -X (n) -AACAGCTCCTCGCCCTTGGACA NN / 3DigN / (only nucleic acids are designated as SEQ ID No.: 214)
[0647] [0647] The modification symbols are those used on the Integrated DNA Technology (IDT) website (5 'DIG = / SDigN /; 3'DIG = / 3DigN /), X (n) means an extension of non-target hybridization nucleotides with indefinite sequence or length (SCNA Connector). Delivery
[0648] [0648] Bioassay configuration: The preparation of the Arabidopsis protoplast is based on Wu et. al. (Wu et. Al., 2009) and is similar to example 1 with differences in the transfection stage: Transfection:
[0649] [0649] The protoplasts are then subjected to FACS or DNA extraction as described below.
[0650] [0650] In this example, cleavage of the target by the molecular complex results in a Double Strand Break (DSB) in the target of the plasmid's DNA. This DSB is created at the PARADA codon site, which is digested and repaired by the endogenous NHEJ repair mechanism. NHEJ is prone to mutations, and some of these mutations can eliminate the STOP codon and restore an open reading frame resulting in an active GFP (ORF) open reading frame. The GFP is then detected by means of microscopy or flow cytometer (FACS), allowing to measure the efficiency of the system and the comparison between variables for its improvement.
[0651] [0651] The efficiency of gene addressing is determined as the percentage of cells positive for GFP. The protoplasts suspended in W5 solution are evaluated
[0652] [0652] The target sequence is a STOP codon coupled to a diagnostic restriction site (Spel ACTAGT, underlined STOP) in the GFP coding sequence. When successfully addressed, the STOP codon and the diagnostic restriction site are eliminated by a deletion, insertion or point mutation event. Repairing a specific condition can also restore GFP expression. The assay is analyzed by FACS as described below or by purifying plasmid DNA from protoplasts using a miniprep plasmid kit (Bioneer K3030) as follows: the protoplasts in the W5 solution are precipitated, and | 250ul of Buffer 1 and carrying out the protocol for bacterial pellets in the manufacturer's instructions. The region between the SCNAs is amplified from the preparation of the resulting plasmid using PCR. PCR products are extensively cleaved with Spel. After electrophoresis, the non-cleaved products are excised from the gel, cloned into a T / A cloning vector (pUC57 / T Fermentas) and individual clones are sequenced to detect different mutation events.
[0653] [0653] To determine optimal SONA distances from potential target sites, for each different target type or type of programmable molecular complex, a group of target plasmids (TARGET-STOPGFP (n), Figure 16) containing a degraded GFP reporter encoding sequence (STOP-GFP) are created. In an artificial N 'leader and in the GFP coding sequence (CDS) two SCNA binding sequences (SCNAbs) are inserted, which flank a target sequence with varying lengths forming a series of plasmids designated as PTARGET-STOPGFP (1- 8) (Figure 16). Inserts are inserted, as outlined in the
[0654] [0654] Other components of the plasmid include 1) a promoter functionally linked to the GFP sequence. The test can be conducted in different eukaryotes. In this example, a plant promoter, such as NosP, is used to conduct the Arabidopsis protoplasty experiment. 2) a pair of SCNA binding sites (SCNA1bs and SCNA2bs); 3) a target site containing a STOP codon; 4) a GFP coding sequence and 5) a terminating transcription sequence, in this example, NosT.
[0655] [0655] The schematic representation (out of scale) shown in Figure 16, illustrates a group of eight exemplary constructs in a group of plasmids pAlvo-STOPGFP (n), containing a reporter coding sequence (STOPGFP) of the fluorescent protein. degraded green (GFP), where “n” means a serial number as shown in the table in Figure 16. The group of inserts of varying length and composition are outlined by an Ncol restriction site encompassing the initial codon and an Mscl site at the end opposite. SCNA1bs is located on the leader N 'artificial GFP and SCNA 2bs is located on the GFP coding sequence. The target sequence is a STOP codon coupled to a diagnostic restriction site (Spel ACTAGT (SEQ ID: 215) or Bell TGATCA (SEQ ID: 216), underlined STOP) and a frame shift (except in n = 5) in the artificial N 'leader. Sequences of target site spacers are shown in example 12. In the table, "n" means the serial number of the plasmid. The distance between SCNAbs in the base pairs (bp) is shown accompanied by the diagnostic restriction site in parentheses. The desired cleavage positions in the upper and lower filaments, due to the 4 projections 5 'of bp, are shown, where +2 numbers are in even-numbered inserts and +3 numbers in odd-numbered inserts, due to the uncertainty caused by the positioning of the catalytic site “on” a nucleotide and not between nucleotides. In some cleavage events, endogenous repair mechanisms can cause imperfect repair, causing deletion, mutation or addition of moldless nucleotides. Some of these repaired sequences can cause the elimination of the STOP codon and the diagnostic restriction site coupled with a shift in the reading frame that restores GFP expression. Minimal restoration events, addition or deletion of nucleotides or point mutations, are shown in the rightmost column of the table. Recognition sequence of the SCNA1 binding in the insert:
[0656] [0656] ATCTCAAGTCTCTAGGACTGGT (SEQ ID: 182) SCNAZ2 binding recognition sequence in the GFP sequence:
[0657] [0657] ATCTGTGAGCAAAGGCGAGGAG (SEQ ID NO: 183)
[0658] [0658] As outlined in Figure 16:
[0659] [0659] Ncol / Mscl insert! for n = 1:
[0660] [0660] COCATGGGATCTCAAGTCTCTAGGACTGGTCTTCAAAATCTTTCTCACTA GTTTCTACGATCTTGGCCA (SEQ ID: 184)
[0661] [0661] Ncol / Mscl insert! for n = 2:
[0662] [0662] CCATGGGATCTCAAGTCTCTAGGACTGGTCAAAATCTTTCTCACTAGTT TCTACGCTGGCCA (SEQ ID: 185)
[0663] [0663] Ncol / Mscl insert! for n = 3:
[06684] [06684] CCATGGGATCTCAAGTCTCTAGGACTGGTAATCTTTCTCACTAGTTACG CTGGCCA (SEQ ID: 186)
[0665] [0665] Ncol / Mscl insert for n = 4:
[06686] [06686] CCATGGGATCTCAAGTCTCTAGGACTGGTAATCTTTCTTGATCAGTCTG GCCA (SEQ ID: 187)
[0667] [0667] Ncol / Mscl insert for n = 5:
[06688] [06688] CCATGGGATCTCAAGTCTCTAGGACTGGTAATCTTTCTTGATCAGCTGG CCA (SEQ ID No: 188)
[0669] [0669] Insert Ncol / Mscl! for n = 6:
[0670] [0670] CCATGGGATCTCAAGTCTCTAGGACTGGTAATCTTTCTTGATCACTGGC CA (SEQ ID: 189)
[0671] [0671] Ncol / Mscl insert for n = 7:
[0672] [0672] CCATGGGATCTCAAGTCTCTAGGACTGGTCTTTCTCACTAGTTCTGGCC A (SEQ ID: 190)
[0673] [0673] Ncol / Mscl insert for n = 8:
[0674] [0674] CCATGGGATCTCAAGTCTCTAGGACTGGTCTTCACTAGTGGCCA (SEQ ID: 191)
[0675] [0675] Each molecular complex is cotransfected into Arabidopsis protoplasts as described below: Delivery
[0676] [0676] Bioassay configuration: The preparation of the Arabidopsis protoplasty is based on (Wu et. Al.) And is similar to example 1 with differences in the transfection step: Transfection:
[0677] [0677] The efficiency of the gene addressing of each form of the molecular complex is tested in the plasmid series pTARGET-STOPGFP (n).
[0678] [0678] When successfully addressed, the STOP codon and the diagnostic restriction site are eliminated by a deletion, insertion or point mutation event (Figure 16). Repairing a specific condition can also restore GFP expression (Figure 16). The assay is analyzed by FACS or by purifying plasmid DNA from protoplasts using a miniprep plasmid kit (Bioneer K3030) as follows: protoplasts in solution W5 are precipitated, and read by adding 250ul of Buffer 1 and performing the protocol for bacterial pellets in the manufacturer's instructions. The "spacer" region is amplified from the resulting plasmid preparation by POR. PCR products are extensively cleaved with Spel (37ºC) or Bell (50ºC) as appropriate. After electrophoresis, non-cleaved products are excised from the gel, cloned into a T / A cloning vector (pUC57 / T Fermentas) and individual clones are sequenced to detect different mutation events.
[0679] [0679] The efficiency of gene addressing is then determined as the percentage of cells positive for GFP. The protoplasts suspended in W5 solution are evaluated for GFP activity 3 days after transfection using an automated flow cytometer (FACS). GFP is detected by excitation at 488 nm with emission detected by a 530/30 filter. Threshold and compensation factors are defined to exclude all false positives.
[0680] [0680] The controls included in the experiment are 1) use of illegitimate SCNAs (non-base pairing) to control non-specific cleavage, 2) use of a PTARGET-STOPGFP lacking a target binding site to control a no action dimer, 3) use of pTARGET-GFP, similar plasmid without the STOP codon for GFP degradation and which has a GFP within the frame as a positive control, 4) use of pTARGET-STOP-I-Scel-GFP, plasmid similar to pTAR-GET-STOPGFP, but containing a | -Scel restriction site near the GFP degradation STOP codon, along with pPSAT4-NLS-I-Scel, a plasmid that expresses a | -Scel restriction enzyme located in nucleus in plant cells, as a control of a comparative heterologous system.
[0681] [0681] The present description regarding the specific modalities will reveal so fully the general nature of the invention that other individuals, when applying the current knowledge, can readily modify and / or adapt these specific modalities for various applications without undue experimentation and without deviating of the generic concept and, therefore, such adaptations and modifications must and are understood in the meaning and scope of equivalents of the revealed modalities. Even though the invention has been described together with specific modalities, it is evident that many alternatives, modifications and variations will be noticed by those skilled in the art. Therefore, the present description intends to embrace all these alternatives, modifications and variations inserted in the essence and in the wide scope of the appended claims. REFERENCES
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权利要求:
Claims (15)
[1]
1. Method for modifying a predetermined target site within a target nucleic acid sequence in a host cell by a programmable nucleus-protein molecular complex, in which the method is CHARACTERIZED in that it comprises releasing to the host cell: a. a programmable polypeptide or a nucleic acid sequence encoding the programmable polypeptide, said polypeptide comprising: (i) a functional domain capable of modifying said target site, the functional domain being deprived of a nucleic acid binding site specific; and (ii) a binding domain that is capable of interacting with a specificity-enhancing nucleic acid (SCNA), the binding domain being devoid of a specific target nucleic acid binding site; B. a specificity-promoting nucleic acid molecule (SCNA), or a nucleic acid encoding SCNA, said SCNA molecule comprising: (i) a nucleotide sequence complementary to a target nucleic acid region; and (ii) a recognition region capable of specifically attaching to the progammable polypeptide binding domain; in which the presence of the polypeptide in the host cell that hosts the SCNA makes it possible to attach said polypeptide to the SCNA, forming an active programmed nucleoprotein complex, thus directing the active programmed nucleoprotein complex to the target site following nucleic acid inside the host cell, enabling the modification of the target site by said active programmed nucleus-protein complex.
[2]
2. Method according to claim 1, CHARACTERIZED by the fact that said target nucleic acid is DNA.
[3]
3. Method according to claim 2, CHARACTERIZED by the fact that said target DNA is genomic DNA.
[4]
4. Method according to claim 3, CHARACTERIZED by the fact that said target genomic DNA is of eukaryotic origin.
[5]
5. Method according to any of the claims | to 4, CHARACTERIZED by the fact that the functional domain is selected from a nucleus, a nickase, a methylase, a methylated DNA binding factor, a transcription factor, a chromatin remodeling factor, a polymerase, a deme - tilase, an acetylase, a deacetylase, a kinase, a phosphatase, an integrase, a recombinase, a ligase, a topoisomerase, a gyrase, a helicase, a double-stranded helix destabilizer, and a Fokl without a specific nucleic acid binding site.
[6]
6. Method according to any of the claims | to 5, CHARACTERIZED by the fact that said target nucleic acid sequence is an extrachromosomal nucleic acid sequence selected from the group formed by mitochondria, chloroplasts, amyloplasts and chromoplasts.
[7]
7. Method according to any of the claims | to 6, CHARACTERIZED by the fact that said target nucleic acid sequence is selected from a viral nucleic acid sequence, a prokaryotic nucleic acid sequence and a synthetic nucleic acid sequence.
[8]
8. Method according to any one of claims 1 to 7, CHARACTERIZED by the fact that said modification is selected from the group formed by mutation, base substitution, deletion, insertion, exchange, binding, digestion, creation of breakage of double tape, nicking, methylation, acetylation, binding, recombination, helical exhalation, chemical modification, marking, activation, inactivation and repression.
[9]
Method according to any one of claims 1 to 8, CHARACTERIZED by the fact that said SCNA comprises a nucleic acid molecule selected from the group formed by a single-stranded DNA, a single-stranded RNA, a double-stranded RNA strand, a modified DNA, a modified RNA, a blocked nucleic acid (LNA) and a peptide nucleic acid (PNA) or combinations thereof.
[10]
10. Method according to any one of claims 1 to 9, CHARACTERIZED by the fact that the interaction between the SCNA and the target nucleic acid occurs through base pairing.
[11]
11. Method according to any one of claims 1 to 10, CHARACTERIZED by the fact that the SCNA recognition region comprises an RNA molecule capable of forming a secondary or tertiary structure.
[12]
12. Method according to any one of claims 1 to 11, CHARACTERIZED by the fact that the association between SCNA modification and said binding domain is a nucleic acid-protein interaction.
[13]
13. Method according to any one of claims 1 to 12, CHARACTERIZED in that the binding domain comprises a polypeptide that binds to a structural RNA motif in the SCNA.
[14]
14. Host cell, CHARACTERIZED by having a predetermined genetic modification at a predetermined target site, created by the method, as defined in any one of claims 1 to 13, wherein said host cell is selected from the group formed by vertebrate, mammalian cell, human cell, animal cell, fungus cell, plant cell, invertebrate cell, nematode cell, insect cell, and stem cell.
[15]
15. Transgenic organism or knock out organism, CHARACTERIZED by having a predetermined genetic modification formed by the method, as defined in any one of claims 1 to 13.
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7. = gi 6 O> 3 "3 222 zZzçº o ss: | $ q 23E à 28: S 3 EE s Se. & 8 eo 3 E 2 oo .-— ã It is $ 3 ana o Ss o 8 a s3s: 3 ss o à: BE S aa E: aa Ss E NE 7 a E is & é S os IA 7 É - 888 fu - s; 3. = = TE and oe a uu 32 & SS Tau is Tee Z 8 and ZA Ss Sos Trans: IF IF. = 2 s3E at $ 5ãà; if ã 8 oo It is the 35DE healthy
Aaj / 8 É) os É Ss A) DTH o uu DA o Ss ÉS to AAA o ERA Ga, 0 PD A In A o = S - Wigiã oo CA Ss 3 o + AA DERA> o> = O 107 2 SA Tá & E É JA 1 Ê õ E Sá E O = SS AND OjE à O DA = * | A 2 É TA TAA Ss Ss o DO O ma Ss 28.65 & EO 2 s8sS'63 o O ERS DE Ss e ovo "o O -c oO = = oo => RA SA O 4th SS << A 2 GIVE oa Pv ESO o 5 ZÉ oO FF 2 og & A 82 3 and 88 S is pu, 7 & Bee ú XX DT IS << = 328 Ne meme aaa and Fq27t CARA O, Ss 8 “TT ey 286 í AO, o - EAN o SS oe RA | E 8R8O O SO | um o E S&S west 5 2 E EE oa 8 PS E à 53 'oS Í E Og
5.0 o S GS oS o is = ”SU ed E Da o o E o E BR = E =! * FM 282 õ <6ço o & z = à = ve O à and SC SS Give = 2 S oO BE eo 8 7 E'S> 2 Ss qo To 3 E <; O o BE E Eis oo EE 3 Are $ 8 o o à o That yours O BS oaoo = É Er = a E Qu mi E oo à 3 Go RR E vo ÃZ a & 382 ”Only EE Ex> 2 os [oo aaa =: £ 3 o 5 | = ss à EE at 27 22º o à DP ma a 38 = ss à à bx GE 2 52 8 to a 1 $ SE O = 8 be Ga 8 o &> Oo m KCENCENTA FÊ 258% BO ã to 6 Ss and S 6 Km Gas o2 = E at c25Ã ”$ om E 85 É | : Ea - 5 2 E ”is GRE << OL A v E E & Ê 2nd o. Â 2 36> 2 ê oo 27070 70
ES º po 2 pa Ç E o 2 = 2 - Or =
O DO VADIA oo o Z & IA AAA ue E Ol EO E Ay fis o Ei Eca 4 ORA = 3 “2 Ee PAG, AGA
DA IS AAA a O 7 io UA CU Ca Nx 2 CIA Link o o Aa = sa o so
EA Z ie | & SÉ is the à o o s = 8 o Ó 8 oO <cs E o at 8 S - o IS F-. <In
PE | vs j E x 332 io 32 3 nm S go Es o vo FS = 3 E So DEE = 7 ss = io) and 3 o = ss = E = Co -> S&S z = 3 Ss zZE = o = OE = PE = = ITA, oo Ko »to EC The PG I gave is A a no = Dr
CTA RIA É ÃO = E mm, Fossa == oo: Fairy
ES ET q
TA Fricá to Es A = | io 8 o. 7 = i o 4 8 m 8 E N 2 5 eh ê F - = ea E ie o <
Cro a, A GL o e E PE DM Ant E a = EMA TA ee 8 Cum cd À Mura Sat 2 o DO - - ADO TE ss 85S = CC mm dd Ri | :: EIS AND CA o o o.
E ii ag> E UU mm) ad 0, 12828 83 GOA ud Ult. $ E5> 2 & ONE ran away at 8 3 80 ox Pe, 1 AA ai o ve 2S E In lo Sn% $ E SS Ei nt ae CS SR SB e É LA eco, BD ud 8 S ã 7 8 8 3 Mm PIA O nto 8 ES E So = SA aa To E Caldo ES esse OO aa da Voo e mA SS ENS Qua oO se SEDES A no A Ca Ed CEA AD Dm, early Ar a E Bee ROS in D o E fo cu ão o inameniD Voss TT po o SEX ME o Ciel E Soo ÃO oa E = A -
E iuis Í——> o = io - 2 E w 2 - o 2 = => - o 8 - o E o in E FO FO is is a and DA
E CAE A E CEO 228 start o ad & TE fu õ E => ot | o E 23 4 = e Eae 8 Ee í = Ss: seo. o o E> 8: so << o: E) o: e + S: o FM 3 o. 2 3 2 so o 2 8 Z 3 o
O 4 te E gs ja KG - E o o no Z = - 8 —— o O = & GADO ES AGA AoA <| ia fam = Pa É CO e O E A OE TRA AM Si Aa, E = É o o Ss É ii Âuuaad CORO o RIAA ARA pr GAARA QD FEAR DO a ATGAARR ÃO ADD BEN AT o A ri - —s fo ie. a |
Á = X o mo "| + Ss o -> oo S .— o [2 ke] o <o 2 o
É A S Ss 8 s 2o = 7 T s and O ions o o o £ Ss o Ss &. oa 8 = & $$ s8PEo: e
BRISLLLIRESS H s2ErET oq no
BESQUESSOS PT Bone Leso - nor esmenoi E o re ET E 8 e Eur s 2 e Fss É o - F &FR; > õ Ss> Ee É em O se c85 O e É HS E 5 SSe, = o ES = S $ ÉES E É SeaXRowos E 8 = Ss, TCB Go í s ESS IS: OA aÉomg o So: E SÉ TURSSES 8 282: o EqEBoEs is mESS AND THESE SSADE 3 Meo I <AOLSOLA 3 IS E ODTea Ta S Foca Ea = S (mà oo es (as e io fds e 08 EL PA Y 22 SEVEN FE OA SE g% o3 Es e AO ST SG ee ". = CA 8 vTÇQESGTE 8 sg A Rm io Elo LevE 5> é = W És ms ge É 2ECETÇÕETNDAO ,. A £ SO CA o 335 EGO E 22 8) CíoLcculoêsmEsno uu 2 2> IF Eh" o = Ss =
Ê S E Ss E so zo = TS o E if R à = sE o Jim IS 2 8 <FAITH »s 65 & É DS A: 88 so | 38 o 12 = Es if 28% õ 22 if ssosts | ES Do go S 2 Eç2 coSQu = | S8E 88 E HE So20 | the eE 2 2 oo Ss, SST A o ECRM oo SS o nO v SS TELSDSO o is EO & SRITSTTRROSu 5 EZ DO Zon dmEL 838 mm NE 8 SST GOXASS << TR fon [é S2 nsces5S Su 3 fo id gr SIZOW DEE Am io Mi Toa TITE DS 2 RA ÃO, ão 8 EE é NS TS um Ê o 14 FANS S 2 E <E Es 8 ke oe & z DA 1 2 E 8 & - E 8 E IF S 85 à o & 32E3 2 SST a Wise SS Soo 25 2LQ3S5 57 = os o SE QE SGELISA E If o. soase = 2bçm 28 gs <Sga8TEES SO MO cr Z <ZZE5SSXZE 8th SsSsSocs Sassi e E 28 E 28 Árkoda> 8EÇrOOo so Ss o 8 & o E o = S TT w E g 8 õ ooo & 2 8 o> E oo £ Ss SE a £ 28 ES o ES oZ Ls AND SE PP o = ns Í 8 8 R 8 O ES Ho a 8 ç To = E 3 = 2 Ss oo = 8 = EsESsçS vs 3 28 o
300002. wo a E mo BE sisso> = Es DES ESSLULCES EE “E = e 2 gm saAZSu Gs - EX S 8 Tm 3 o NxÃA A E E ZEZL SS 2 at OEZ "6S“ "4 é = £ S ê Re THE WEIGHT 2> 2 fia, TOS OD 3 = = Joá ici =; E 8 + o a a:; s 2 o 58 SE = À E É SS SGogs O na TA OE THIS use P <s & 17: / dJ $ "PFSTISHSOS É = FxXoGo TS o QE = / 12A 2 B $ S8TSATZ fo a 2/0 SS oOnsgESA a é Âã Dida | DRL2QEGEOS Ss Gm Um 382 uUR2QELT EOE VA EeEcí, aEoSTES o ÃO, TECESIHCPIPCES Ss FARRA E. SARA = Dil 0230 Ss AGR 7 A AZSAPICOAAL js for SA 8 AR F "- 8 2 Es PRA "Se. Oo io se Ss 3 x Êo, sm 28 Duas + 2 - É Es BLESS So 9 8 oa) are SS and ns 8 8 9 ão 2 = 2nd g & Ssssasasas | so | 8 cQ 0078 8 mo 3ZoeNnNCC dm if Ss 882 E S2o5maesisos Bo EE um ns CELCXLZOUFRL e ss E Bia aaa EE = = E DO í OE RX and ÃO e = Fi PEA = Bs E us Ss 8 gre P 38 bd E A sE2 o Ss Es 8 ss 8) = the $ 3 ao É o0- O É às É Ses << Faves A x 8 5 28X E sSSs És ss (À & D 1.8 Soo 25220365 2 E iso 2ocESECDOÕE 83 ”2228005 8 ESHTOOS EX) ESASSOSSSA cex <A <L BOEITFZQuUNOUERISAZ SonpnréuwoX> O6rFroo CEO O TC Lo Aa a ada ea so Nm Se E.
NE & | It is 5 Ss a> DEN o is i = 8 à = & E ST a, un & t HM * m E is Oo ”E E Sta.
NO AAA 8 of .. S o St.
E = o & e | E = E a) and <<| 63 to Z Ss ol> o o) | is AND —Em.
At kilos ms o =; (=) 38 E Ss E | 8 and ol o = / | 23 | o 6 & a o no e “A 2a e go E E” / ao VA q e —fM 8 b: on o i 2oO: s2 f,: sm É i & í ht i * í A Q i mo | ml ue o e x O / | SO A - ol 8 | ui IS 1 "À E | oa E o 8 pray <S 2 mr 2 5. s vorão sã E To = 0 o ho à. | e [o | BO o - E 1 ÍE EE à = ES SA eis tn DO e fointao: E & << ee SS wo> EE = [s GEEXR O 2 o BP lazco 3 s 8 18 TEA 2) and o = AAA spindles F oe 6) and Md ir E Ss Ss and Tm Ti - Í E ã COS AA - lh>, o à 5) 2 E SN hj S e <e a OS) Ff y 2 Vi // & | io: O Rc En FA + PA = A - A. 2 E = - Do A = [e Sã CR AÁ / = Mo 55 Fm ss PDA DA = EmE .. a im = CORA = WO SS HA 85 EE ud = EF 8 S AR = ES | 8 No = PE e.
S S Hm E E o = FPP. sv ÀS | of ATA = 15 m and S g E »Ê 8 = 8 '| % NT EA =) 8 It is 5 o.
E 3 E É g D = 3 Ss 2 is NEI: 2— q. = - And 8 Ma =. | E =: g E 2 o Ts a = ao 23 AA - E 8. E A = W ES EO BO = UM Ss SS Ã »EN im o E a.
AAA o HW A = Jo, ZA & VS EA A s 1 o W = 5 AS $ À) a dO N> VIM 8 already <Í s A o à o É = do S o HZ | a EO 2 Aa HRS bread m "E DP AA, V E e is e. 2 eo e É E = | ad
Ss E E = 1 = | 2 = = | 7 OS Es. À e WS 122 NA Fa o F o BREAD o = SS EA S & a EU õ already Va> ZE = + ss Ê 1 sr oE = 0> fem << (vain To oE = 5 8> San 2 O = ã E 8 IF 2 z and N 2 ==) 3 2 << and E = 3 if> at = 2 = TF z ss O 8 Nim Fico: Ê í z Ao $ A & Ho EE ES pi, ã so <LO AND FF 2 o ORA A oo ê = 4% S 2 FD = EE À & S fm 4 S POA A a <us DR AS: DD 5
SÉ Â ZE only * s 2 = oz TEA O o g TA E <| Are you - & [Ko ir TiZ 13 are £: (a) sEZS: 22 O 2 FS 22 = e Ti à = PD 8 Aa 8X í Tb er <€ <o Po Po new SAO 1 1: 2Ê & DO ei to FE S 8 Z% Es o Fi is% | A o "o 8 o 2 o * Ss BO o pl Ss 5 z> 3 IS FE FAIRY, o s <W mM s o <Ss o 3 E: 2 O
AND
Ss AA o gs E gi PÃO ns * ET O E E and Pd É Es be ES Z2 O .o Es n à. "ÃO o ZzE = .: - í IS 8 SE = 2 s DOE & == o [ANS 4 <E = 8 g it <22 ZE = 32
2. o = 8 2 Z 8. FE.
Fi so MD e = S o ss = - o ace EZ Nm E | the ss FRA, the: == <as. "ae E« o = f o.> Ped EO o To)% iz ús FRA A o GO, [RA o - a ES ARAGÃO TE) ES E CO, Ens BAGA ATA Eis ss G LD E mo sie) * s8 1 »ETA A -> 2% o FR E AAA o ls% ão A» IS CA S&S Magnet W AO = CA 22 mm € E ”. Â EE Bm E 4 izizã 2 Ss ss F ão = E =:% the DD sm Ss o mãa o 2 o <PA sz 3 2 FP == Ss EZX E fa -ZÉHO e = = (Er oe AN gua $ É go 4 mom & é e o ai ai cm cm Ê sm o E EA N ki EE the GOA DES A EO q. EE DT oo IS 3 EE 1. 3 as E i ". Ê Ã eí% E- 2 E Cv | FE“ PS AS -] ES 2 & a 8 = a and wo GIVE rr the st 2nd SS give 3 S o *% vp SS ÃO Ss 8 & 2 8 E 3 Ss & E% o ão & 2 ”and 2 ge ooa É Es do Um ss” nm ES <E m 1 35 AS co - 8 ER ". (E E = o T 2 dk 3 & E za <$ Z oz õ $ 5e o E% AA, oÓ o = = ÃO A o DO 28 FA 3 =: - mB = 2 S a E Edo e 5 = õ 5 re 1 SS = e = poa E == O q AS OS e À = es 2 IS 2 o FETUS 2 o E & 2 AO o $ A = - ao 2 e = E ÃO 7 dA == E NS 2 e É * e o Tem vo PN) 1 DA = a FRO H SUM 38 HS: TPR ae E 08 DD (iq> PG R "1 PEN is O JJ = Pi ad a ã 5 EA a E Ss + o MM <ro = E VS ó A = ê A, “" x 5 O Ss o s 1 W oo i L = ã EE | A, sá 1 vo 26 i v 5 o FF j 2 E 8 à; E 88 E j £ 88 j o o 7 8: o Í 2% o o 9 2 o z E.
S o u sS So o.
As part o 5 o e A 2 3 co = dA HA 3 Ss o 8 - E 2 2 = 2 & $ o 2 ê ce E rs 3% 8 In 8 ú o = s id o; o - S $ 8 38. = Es o cs Ed Õ-s 2 me = o GIVE 2 SPECT ss ss É = 288 SrTgito ns = faith ES XT ES s Á o Im 2ºno SEE EO = & LA EO; oS82E 2E = se It's 2nd &. There are 1 videos Or Z o ST:; çE 1 FE SSL TEN IF Am is Bos HZ oEcS = << fa ”Rss ÍgFoSS fa = = = [PT 221 ÁÂÁ owEos É rs on DE Zva F2TESsSB IS ES h Am 23 TZ <20 VA A o so2o << ã Cm md SS 325 = To Vaz) In ema (o E Milo ADS O TR 23 q 2): AM 4 3 Toa) 22 QB 2 to ss & SO EE 2 22 ES CEO 2 Eh = = E. ss 2 = 3 £ Az Ss is 23oO EA = Z 8 si jo ÃO = í s jo g "ã ã zX 2 8 C qoo Ô oo 8 o> oz S sv É 8 o EE 3 o õ e 8 < o É s <<8: ooo 3 ns o 2 oO - ao = mm os i à o FAITH ij a 2 8 o = e 8 = Dei: ã ze: ã go ã 26. á S. à 2 8º Z É ma : SE ser u s E s = ES S mA s s 2 gr: Sos E 2º *: Es = 2 We Ss ia = Ô PAGÃO, SEN 2 fins = 9 =) “trt. E 3: 38º $ 0: / 27 > É Eb BIA, É É E E PD É Ea E Ó 4 É poa tt CS Ex es A o ao la IS = AA - à is fans à Á 2m RE IT'S HIM n TEA eo É 3 3 EO, ÉS = 3rd AR E = E Ms H> = = IS OS IS O 2 e * À = NHS g: À 8 3 “É 8. À 8 BB E * CS z - ET É 8 a sz 2 Be 2 83 <3 so < 2 É so x É oO É = É To 4 É E> É SS 2 os ÁEoo
It's DOF Pas úgGeo Za ho E2PÉIA * nego 7 ho 1! ES o: 3. 18 8 «SS = 8 SS 1282 makes the OE 3“ WIRE. lI8BB iSTE -./385s5 P iFRgo IS 200 nNeO
" The ; ss à "Su82 io a ves | e 18 2 8a Z 1i3ã THERE ARE E AND E IS. ss q IS” ES à E dom & se Á = A Ípas Es o so o = A * nom = AA Ss ND Adam 2 -2 E 122 is RE ZE 8 1:. S85E OEsS £ 8 ki. 2582 PNEZX 3 o dO, = “úseãs a N / A 8 AA» FEZ "& = as day, Elm FAN H 2 CA to H 2 e f G . healthy IS 5; A A, x: DOG = 2H 2H IS == 2z BS = = A IS S&S 27 Ez ÉS = 279 =: At s 2 o = e = À H z 2 É IS FR a 2 E 8 s 8 | À H ov 7 Hs S 2 8 ú E: S 3 & ú ST = E% s O z 1 É & Ss = 2 "Es = 7 q S" és <5 and 4 rá 23 E <e Ss Hs EE SS . 3rd is ”= 752 | :s . 2 ZE Õ S Es 2.> A É É oo 8 x OE KA ms mol Ss a SSE & ENE o E g% E 3 2 sss So there is PE DO <> ii SSL PA ii: BSS É Ci Res 4 CBR | Z ii Et = a FF 8 sSã | = E “FR S is 2 2 IS - o 8
É and dj é É é Ss É 's 21 8 Pa Sou: E Wool E ses:; ZES Z-á> 08 ú Ss Res p = 32, É and Pa at 25, á = EM so: e É 2 Ao E (os Has: si 2 AE E ja As EE A os ER r2o, q 8 / 4 FOOT ZE, Ds IS,> E ES 272 2: É do Vo E SS E 8 3 io ae = é. Af CO Ss PP. Sound: AE e) E mo SU cs = & àa2s' Sá 9: It is the 2nd as PS for AIDS and the 1st as Eos q ss Ah O |. S&S ie.
From E: ii, ss SEA or E: dos es ==. INTE S 4) o o = x to ERAS o 7 at o: if E Mon AND 8 SEE IS WILL HAVE E.
FE E 1 AND 3 os E = s AND VV E E 4 8 Es E 26 & E 5 ã | > “E Í EA ã F 58 á <7 ses É & o 8 ã Rs 2 ã ú% É É E s ã It is FN í É aj is healthy à Z ã 2 s É 3 É 2 z.ã 2º sm É x mi po OE E e 8 <q = E E GA GA EO: E WE õ E EU.
Ss dh: 8 1a Ss & E Os 0: 6 d o / A ED. = 007: Ss E SS s / D & É Ó fm dd SW PP o Y sa Dc DA o E: ms í Ss 7 É ad do 2 É g SS HE WI É É oo Bo = = oiÉ Ss 2 os e 2EIS Fo 8 go & SE dido 8 pin and 2 oo 25H 2 & fe, SS o eo <Ss is Ss s = 3 = & S 8 & <2 = ok oj É 2 i ai AND EE É So = 2 = = ES BO oo ÃO , = um =) and DÃO É fade) SR, = AZ XY CE SRA 48> use CS = (2% CP É & ad À ==) À É Éo EA = E o> A 3 [= | o H e É = À E = o À S c SH & 285 Z 8 É SA u cs e Eu 8 'É Eco) o 8 & => 268 ”ks) E Woo o É Áx 2 s 9 Hs Z28 <E o ZE TRF VV F 225 õ Z 3 s * É PE 2 é 8 3 8 É F << 4 o BP = = <It is in Z-s OEs AA & s OZ Ss X O É Zz = O <a 4 É zo Cad CA MADE one: CO 2 o 1 o ÃO ZE -; is WEZ> =) GA FE EE = 3 El = 2 ES o À only 3 = Déo <so <. =
AND

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法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161576423P| true| 2011-12-16|2011-12-16|

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US61/576.423|2011-12-16|

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PCT/IL2012/050528|WO2013088446A1|2011-12-16|2012-12-16|Compositions and methods for modifying a predetermined target nucleic acid sequence|

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