ENDONUCLEASE, METHOD FOR HOMOLOGICAL RECOMBINATION OF POLINUCLEOTIDES AND METHOD FOR DIRECTED POLINU

专利摘要:
endonuclease, method for homologous polynucleotide recombination and method for directed polynucleotide mutation. These are optimized endonucleases, as well as methods of targeted integration, targeted deletion, or targeted mutation of polynucleotides using optimized endonucleases.
公开号:BR112012012588B1
申请号:R112012012588-5
申请日:2010-11-25
公开日:2019-03-26
发明作者:Andrea Hlubek；Christian Biesgen
申请人:Basf Plant Science Company Gmbh；
IPC主号:

专利说明:

“ENDONUCLEASE, METHOD FOR HOMOLOGICAL RECOMBINATION OF POLYNUCLEOTIDES AND METHOD FOR DIRECTED MUTATION OF POLINUCLEOTIDES”
Field of the Invention [001] The present invention relates to optimized endonucleases, as well as methods of targeted integration, targeted exclusion or targeted mutation of polynucleotides that use optimized endonucleases.
Background to the Invention [002] Genomic engineering is a term to summarize the different techniques for inserting, deleting, replacing or otherwise manipulating specific genetic sequences in a genome and has several therapeutic and biotechnological applications. Almost all genomic engineering techniques use recombinases, integrases or endonucleases to create double stranded DNA breaks at predetermined sites in order to promote homologous recombination.
[003] Despite the fact that several methods have been employed to create double stranded DNA breaks, the development of effective means to create double stranded DNA breaks at highly specific sites in a genome remains a major goal in gene therapy , agrotechnology, and synthetic biology.
[004] One approach to achieve this goal is to use nucleases with specificity for a sequence that is large enough to be present only at a single site in a genome. Nucleases that recognize such large DNA sequences of about 15 to 30 nucleotides and are therefore referred to as "meganucleases" or "addressing endonucleases" and are often associated with parasitic or redundant DNA elements, such as introns and inteins from
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2/71 auXo-splicing of group 1 commonly found in the genomes of plants and fungi. Meganucleases are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural reasons, which affect the catalytic activity and the sequence of their DNA recognition sequences.
[005] The natural meganucleases of the LAGLIDADG family have been used to effectively promote site-specific genomic changes in insect and mammalian cell cultures, as well as in many organisms, such as plants, yeast or mice, however, this approach is limited to the modification of its homologous genes that conserve the DNA recognition sequence or genetically pre-modified genomes in which a recognition sequence was introduced. In order to avoid these limitations and promote genetic modification stimulated by a double stranded DNA break, new types of nucleases were created.
[006] One type of new nucleases consists of artificial combinations of non-specific nucleases to a highly specific DNA binding domain. The effectiveness of this strategy has been demonstrated in a variety of organisms that use chimeric fusions between a genetically engineered zinc finger DNA binding domain and the non-specific nuclease domain of the Fokl restriction enzyme (eg example, WO03 / 089452), a variation of this approach is to use an inactive variant of a meganuclease as a DNA binding domain fused to a non-specific Fokl-like nuclease as described in Lippow et al., “Creation ofa type IIS restriction endonuclease with a longrecognition sequence ”, Nucleic Acid Research (2009), Vol.37, No.9, pages 3061 to 3073.
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3/71 [007] An alternative approach is to genetically engineer natural meganucleases in order to customize their DNA binding regions to link existing sites in a genome, thus creating genetic engineering modified meganucleases that have new specificities (for example, W007093918, W02008 / 093249, WO09114321). However, many meganucleases that have been modified by genetic engineering in relation to the specificity of DNA divage have decreased divage activity in relation to the naturally occurring meganucleases from which they were derived (US2010 / 0071083). Most meganucleases also act on sequences similar to their optimal binding site, which can lead to unintended or even detrimental off-target effects. Several approaches have already been adopted to increase the efficiency of homologous recombination induced by meganuclease, for example, fusing the nucleases to the Glucocorticoid Receptor ligand binding domain in rats in order to promote or even induce the transport of this modified nuclease to the cell nucleus and, therefore, its target sites through the addition of dexamethasone or similar compounds (W02007 / 135022). Despite this fact, there is still a need in the art to develop meganucleases that have high rates of homologous recombination induction and / or a high specificity to their binding site, thus limiting the risk of off-target effects.
Brief Description of the Invention [008] The invention provides optimized versions of endonucleases from the LAGLIDADG endonuclease family. In particular, optimized endonucleases comprise an amino acid sequence that has at least 80% amino acid sequence identity to a polypeptide described by SEQ ID NO: 1, 15, 16, 17 or 19. In one embodiment of the invention, endonucleases optimized versions are wild-type or
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4/71 modified by l-Scel genetic engineering, as described by SEQ ID NO: 1 or one of its counterparts that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity, which has one or more mutations selected from the groups of:
a) l-Scel -1, l-Scel -2, l-Scel -3, l-Scel -4, l-Scel -5, l-Scel -6, IScel -7, l-Scel -8 and l- Scel -9;
b) S229K, S229A, S229P, S229G, S229E, S229Q, S229D, S229N, S229C, S229Y, S229T, M203K, M203H, M203R, Q77K, Q77H, Q77R, E130K, E130H19, E1
c) a methionine, valine, glycine, threonine, serine, alanine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, isoleucine or histidine after the methionine starting from its amino acid sequence; or
d) a combination of one or more mutations selected from a) and b), a) and c), b) and c) or a) b) and c) above.
[009] In an embodiment of the invention, the optimized endonuclease comprises an amino acid sequence described by SEQ ID NO 2, 3 or 5.
[010] In a further embodiment of the invention, optimized endonucleases are a genetically engineered version of an endonuclease that comprises an amino acid sequence that has at least 80% amino acid sequence identity to a polypeptide described by SEQ ID NO: 1, 15, 16, 17 or 19.
[011] In another embodiment, the invention provides an endonuclease that has at least 80% amino acid sequence identity to a polypeptide described by SEQ ID NO: 1, or a genetically engineered version of an endonuclease that has at least 80 % amino acid sequence identity to a polypeptide
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5/71 described by SEQ ID NO: 1, wherein the amino acid sequence TISSETFLK is removed by deleting or mutating any of the amino acids in the amino acid sequence TISSETFLK. Another preferred embodiment of the invention consists of an optimized endonuclease according to any one of claims 1 to 4, comprising an amino acid sequence having at least 80% amino acid sequence identity to a polypeptide described by SEQ ID NO: 1 or 2 and comprises a serine Nr 229 mutation of SEQ ID NO: 1. In a further embodiment of the invention, the optimized endonuclease is fused to at least one zinc finger domain, or at least one unit of zinc finger. repetition derived from a transcription activator-type effector (TAL), or at least zinc finger domain (zinc finger) and at least one repeat unit derived from a transcription activator-type effector (TAL). Preferably, the optimized endonucleases comprise a Seclll or SecIV secretion signal. The invention also provides isolated polynucleotides comprising a polynucleotide sequence, which codes for an optimized endonuclease.
[012] Preferably, this polynucleotide is codon optimized or has a low content of RNA instability motifs or has a low content of codon repetitions, or has a low content of cryptic splicing sites, or has a low content of codons alternative initiation sites, have a low content of restriction sites, or have a low content of secondary RNA structures or have any combination of the characteristics described above. Another embodiment of the invention consists of an expression cassette comprising an isolated polynucleotide as described in functional combination with a promoter and a terminator sequence. Other embodiments of the invention are vectors, host cells or non-human organisms comprising a polynucleotide encoding an optimized endonuclease, or an isolated polynucleotide encoding an optimized endonuclease, or an
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6/71 expression cassette comprising a polynucleotide encoding an optimized endonuclease, and vectors, host cells or non-human organisms comprising a combination of the endonucleases, polynucleotides and expression cassettes described above. Preferably, the non-human organism consists of a plant.
[013] The invention provides methods of using the endonucleases described herein to induce homologous recombination or terminal bonding events, preferably in methods for targeted integration of sequence excision. Preferably, the sense sequences excised are marker genes. The invention further provides a method for homologous recombination of polynucleotides comprising the following steps: a) providing a competent cell for homologous recombination, b) providing a polynucleotide comprising a DNA recognition site of an optimized endonuclease flanked by an sequence A and a sequence B, c) providing a polynucleotide comprising sequences A 'and B', which are long enough and homologous to sequence A and sequence B, to allow homologous recombination in said cell and d) providing an endonuclease optimized as described in this document or an expression cassette as described in this document, e) combining b), c) and d) in said cell and f) detecting the recombined polynucleotides from b) and c), or selecting or culturing cells comprising recombinant polynucleotides b) and c). Preferably, the method for homologous polynucleotide recombination leads to homologous recombination, with a polynucleotide sequence comprised in the competent cell in step a) being excluded from the genome of the cells in cultivation in step f). A further method of the invention consists of a method for targeted mutation which comprises the following steps:
a) providing a cell that comprises a polynucleotide that comprises
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7/71 an optimized endonuclease DNA recognition site, b) providing an optimized endonuclease according to any one of claims 1 to 7 or an expression cassette according to claim 10 and which is capable of cleaving the site of DNA recognition from step a), c) combining a) and b) in said cell and d) detecting mutated polynucleotides, or selecting or culturing cells comprising mutated polynucleotides.
[014] In another preferred embodiment of the invention, the methods described above comprise a step, where the optimized endonuclease and the DNA recognition site are combined in at least one cell through crossing organisms, through transformation or through a transport mediated via a Sec III or SeclV peptide fused to the optimized endonuclease.
Brief Description of the Figures [015] Figure 1 shows a comparison of the frequency of homologous recombination, measured by the restoration of beta glucuronidase activity (% of blue seedlings), after recombination induced by three different l-Scel variants. Each l-Scel variant was tested on five different plant strains, leading to test construction. For each combination, 96 seedlings of generation T2 were analyzed to determine beta glucuronidase activity (“l-Scel”, which has the amino acid sequence described by SEQ ID NO: 1; “l-Scel c-term mod” which has the amino acid sequence described by SEQ ID NO: 3; "NLS IScel c-term mod", which has the amino acid sequence described by SEQ ID NO: 5), see also Example 10b. Figure 2 describes a sequence alignment of different l-Scel homologues, where 1 is SEQ ID NO: 1, 2 is SEQ ID NO: 15, 3 is SEQ ID NO: 16, 4 is SEQ ID NO: 17, 5 is SEQ ID NO:
18.
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Detailed Description of the Invention [016] The invention provides optimized endonucleases, which can be used as alternative enzymes for inducing double stranded DNA breakage. The invention also provides methods of using three optimized endonucleases.
[017] Optimized endonucleases are variants of ISce-l (described by SEQ ID NO: 1) and of ISce-l counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity. The optimized versions of l-Scel are also referred to as ISce-l optimized.
[018] ISce-1 endonuclease homologs can be cloned from other organisms or can be created by mutating LAGLIDADG endonucleases, for example, by substituting, adding or excluding amino acids from the amino acid sequence of a determined LAGLIDADG endonuclease.
[019] For example, it is possible to add nuclear localization signals to the amino acid sequence of a LAGLIDADG endonuclease and / or change one or more amino acids and / or exclude parts of its sequence, for example, parts of the N-terminal or parts of its C-terminal.
Table 1: Exemplary counterparts of I-SceI, which can be cloned to
FROM OTHER ORGANISMS DESCRIBED IN TABLE 1;
Accession No. Uni-Prot Body SEQ ID NO 1 amino acid sequence identity A7LCP1 S. cerevisiae 1 100 Q36760 S. cerevisiae 15 98 063264 Z. bisporus 16 72 Q34839 K. thermotolerans 17 71 Q34807 P. canadensis 18 58
[020] The LAGLIDADG endonucleases useful in the invention can be found in the genomes of algae, fungi, yeasts, protozoa,
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9/71 chloroplasts, mitochondria, bacteria and archaea. LAGLIDADG endonucleases comprise at least one conserved LAGLIDADG motif. The motif name LAGLIDADG is based on a characteristic amino acid sequence that appears in all LAGLIDADG endonucleases. The term LAGLIDADG is an acronym for this amino acid sequence according to the one-letter code as described in STANDARD ST.25, that is, the standard adopted by the PCIPI Executive Coordination Committee for the presentation of nucleotide and amino acid sequence listings in patent applications.
[021] However, the LAGLIDADG motif is not completely conserved in all LAGLIDADG endonucleases, (see, for example, Chevalier et al. (2001), NucleicAcids Res. 29 (18): 3757 to 3774, or Dalgaard et al. (1997), Nucleic Acids Res. 25 (22): 4626 to 4638), in such a way that some LAGLIDADG endonucleases comprise some or several amino acid changes in their LAGLIDADG motif. LAGLIDADG endonucleases comprising only one LAGLIDADG motif generally act as homodimers or heterodimers. LAGLIDADG endonucleases comprising two LAGLIDADG motifs act as monomers and generally comprise a pseudo-dimeric structure.
[022] LAGLIDADG endonucleases can be isolated from polynucleotides of organisms mentioned as examples in Table 1, or synthesized again by techniques known in the art, for example, using sequence information available in public databases known to persons skilled in the art, for example, Genbank (Benson (2010)), NucleicAcids Res38: D46-51 or Swissprot (Boeckmann (2003), Nucleic Acids Res 31: 365-70). A collection of LAGLIDADG endonucleases can be found in the PFAM Database for protein families. The accession number of the PFAM Database PF00961 describes the LAGLIDADG 1 protein family, which comprises about 800 protein sequences. O
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10/71 Accession number of the PFAM Database PF03161 describes members of the LAGLIDADG 2 protein family, which comprises about 150 protein sequences. An alternative collection of LAGLIDADG endonucleases can be found in the InterPro database, for example, InterPro accession number IPR004860.
[023] Another way to create LAGLIDADG endonuclease homologs is to mutate the amino acid sequence of a LAGLIDADG endonuclease in order to modify its DNA-binding affinity, its dimer-forming affinity or to alter its DNA recognition sequence. Determining the structure as well as the sequence alignments of LAGLIDADG endonuclease homologs allows rational choices regarding amino acids that can be altered to affect their DNA binding affinity, their enzymatic activity, or alter their DNA recognition sequence.
[024] Depending on the usage in question, the term “DNA binding affinity” means the tendency of a meganuclease or LAGLIDADG endonuclease to associate non-covalently with a reference DNA molecule (for example, a DNA recognition sequence or an arbitrary string). The binding affinity is measured by a dissociation constant, KD (for example, the KD of l-Scel for the WT DNA recognition sequence is approximately equal to 0.1 nM). Depending on the use in question, a meganuclease "altered" the binding affinity if the KD of the recombinant meganuclease for a reference DNA recognition sequence was increased or decreased by a statistically significant amount (p <0.05) over a reference meganuclease or a LAGLIDADG endonuclease.
[025] Depending on the usage in question, the term “enzyme activity” refers to the rate at which a meganuclease, for example, an endonuclease
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LAGLIDADG cleaves a particular DNA recognition sequence. This activity is a measurable enzymatic reaction, which involves the hydrolysis of double-stranded DNA phosphodiester bonds. The activity of a meganuclease that acts on a particular DNA substrate is affected by the affinity or avidity of the meganuclease for that particular DNA substrate which, in turn, is affected by both sequence-specific and sequence-specific interactions with DNA.
[026] Nucleases can be further optimized by excluding 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids from their amino acid sequence, without destroying their endonuclease activity. For example, if parts of the amino acid sequence of a LAGLIDADG endonuclease are excluded, it is important to retain the LAGLIDADG endonuclease motif described above.
[027] It is preferred to exclude PEST strings or other reasons for destabilization such as KEN-box, D-box and A-box. These motifs can also be destroyed by introducing unique amino acid exchanges, for example, introducing a positively charged amino acid (arginine, histidine and lysine) in the PEST sequence. LAGLIDADG endonucleases, which have been mutated for the purpose of modifying their DNA binding affinity, or altering their DNA recognition sites are termed as genetic engineering modified endonucleases. I-Scel, as well as l-Sce I counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% of amino acid level sequence identity can be genetically modified like other LAGLIDADG endonucleases in order to alter their DNA binding affinity, their enzymatic activity, or alter their DNA recognition sequence . Genetically engineered versions of l-Scel and l-Sce I counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94% ,
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95%, 96%, 97%, 98% or 99% amino acid level sequence identity.
[028] Correspondingly, in an embodiment of the invention, the optimized endonucleases are versions modified by genetic engineering of l-Scel or their counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85% , 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity at the amino acid level and which has an altered DNA binding affinity, an altered enzymatic activity , or an altered DNA recognition sequence, when compared to its unmodified form by genetic engineering, meaning the respective LAGLIDADG endonuclease as it occurs in nature.
[029] In another embodiment of the invention, the optimized endonucleases are variants of l-Scel as described by SEQ ID NO: 1 or their counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85% , 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acid level sequence identity as they occur in nature.
[030] Homologues, which do not occur in nature, but have at least one of the mutations A36G, L40M, L40V, 141 S, 141 N, L43A, H91A and I123L, which have a small effect on the DNA binding affinity of IScel, or will alter its DNA recognition sequence, will also be considered to be homologous that occur in nature, as long as they do not understand other mutations, which alter their DNA binding affinity, their enzymatic activity, or their DNA recognition sequence, when compared to l-Scel as described by SEQ ID NO: 1 or its respective counterpart that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% amino acid level sequence identity as occurs in nature. Genetically engineered versions of l-Scel, which have an increased DNA binding affinity or
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13/71 reduced are, for example, described in WO07 / 047859 and WO09 / 076292 included herein by reference.
[031] If not explicitly stated otherwise, all mutants will be named according to the amino acid numbers of the wild type amino acid sequences of the respective endonuclease, for example, the L19 mutant L-Scel will have an amino acid exchange of leucine at position 19 of the wild-type l-Scel amino acid sequence, as described by SEQ ID NO: 1. The L19H mutant of l-Scel will have a substitution of the amino acid leucine at position 19 of the wild-type l-Scel amino acid sequence with histidine.
[032] For example, l-Scel DNA binding affinity can be increased by at least one modification corresponding to a substitution selected from the group consisting of:
(a) replacement of D201, L19, L80, L92, Y151, Y188, 1191, Y199 or Y222 by Η, N, Q, S, T, K or
R; or (b) replacement of N 15, N 17, S81, H84, N94, N 120, T156, N 157, S159, N163, Q165, S166, N194 or S202 with K or R;
the DNA binding affinity of l-Scel can be reduced by at least one mutation corresponding to a substitution selected from the group consisting of:
(a) replacement of K20, K23, K63, K122, K148, K153, K190, K193, K195 or K223 with Η, N, Q, S, T, D or E; or (b) replacement of L19, L80, L92, Y151, Y188, 1191, Y199, Y222, N15, N 17, S81, H84, N94, N120, T156, N157, S159, N163, Q165, S166, N 194 or S202 by D or E.
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14/71 [033] Genetically engineered versions of l-Scel, l-Crel, l-Msol and l-Ceul that have an altered DNA recognition sequence are described, for example, in WO07 / 047859 and WO09 / 076292.
[034] For example, an important l-Scel DNA recognition site has the following sequence:
sense: 5'-TTACCCTGTTATCCCTAG-3 'basic position: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 antisense: 3'-A AT G G G A C A AT AG G G AT C-5'.
[035] The following mutations of l-Scel will change the preference for C in position 4 to A: K50.
[036] The following mutations of l-Scel will maintain the preference for C at position 4: K50, CE57.
[037] The following mutations of l-Scel will change the preference for C in position 4 to G: E50, R57, K57.
[038] The following mutations of l-Scel will change the preference for C in position 4 to T: K57, M57, Q50.
[039] The following mutations of l-Scel will change the preference for C in position 5 to A: K48, Q102.
[040] The following mutations of l-Scel will maintain the preference for C in position 5: R48, K48, E102, E59.
[041] The following mutations of l-Scel will change the preference for C in position 5 to G: E48, K102, R102.
[042] The following mutations of l-Scel will change the preference for C in position 5 to T: Q48, C102, L102, V102.
[043] The following mutations of l-Scel will change the preference for C in position 6 to A: K59.
[044] The following mutations of l-Scel will maintain the preference for
C in position 6: R59, K59.
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15/71 [045] The following mutations of l-Scel will change the preference for C in position 6 to G: K84, E59.
[046] The following mutations of l-Scel will change the preference for C in position 6 to T: Q59, Y46.
[047] The following mutations of l-Scel will change the preference for T in position 7 to A: C46, L46, V46.
[048] The following mutations of l-Scel will change the preference for T in position 7 to C: R46, K46, E86.
[049] The following mutations of l-Scel will change the preference for T in position 7 to G: K86, R86, E46.
[050] The following mutations of l-Scel will maintain the preference for T at position 7: K68, C86, L86, Q46 *.
[051] The following mutations of l-Scel will change the preference for G in position 8 to A: K61, S61, V61, A61, L61.
[052] The following mutations of l-Scel will change the preference for G at position 8: E88, R61, H61.
[053] The following mutations of l-Scel will maintain the preference for G at position 8: E61, R88, K88.
[054] The following mutations of l-Scel will change the preference for G in position 8 to ο Τ: K88, Q61, H61.
[055] The following mutations of l-Scel will change the preference for T in position 9 to A: T98, C98, V98, L9B.
[056] The following mutations of l-Scel will change the preference for T in position 9 to C: R98, K98.
[057] The following mutations of l-Scel will change the preference for T in position 9 to G: E98, D98.
[058] The following mutations of l-Scel will maintain the preference for
T in position 9: Q98.
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16/71 [059] The following mutations of l-Scel will change the preference for T in position 10 to A: V96, C96, A96.
[060] The following mutations of l-Scel will change the preference for T at position 10 to C: K96, R96.
[061] The following mutations of l-Scel will change the preference for T in position 10 to G: D96, E96.
[062] The following mutations of l-Scel will maintain the preference for T at position 10: Q96.
[063] The following mutations of l-Scel will maintain the preference for A at position 11: C90, L90.
[064] The following mutations of l-Scel will change the preference for A in position 11 to C: K90, R90.
[065] The following mutations of l-Scel will change the preference for A in position 11 to G: E90.
[066] The following mutations of l-Scel will change the preference for A in position 11 to T: Q90.
[067] The following mutations of l-Scel will change the preference for T in position 12 to A: Q193.
[068] The following mutations of l-Scel will change the preference for T in position 12 to C: E165, E193, D193.
[069] The following mutations of l-Scel will change the preference for T at position 12 to G: K165, R165.
[070] The following mutations of l-Scel will maintain the preference for T at position 12: C165, L165, C193, V193, A193, T193, S193.
[071] The following mutations of l-Scel will change the preference for C in position 13 to A: C193, L193.
[072] The following mutations of l-Scel will maintain the preference for
C at position 13: K193, R193, D192.
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17/71 [073] The following mutations of l-Scel will change the preference for C in position 13 to G: E193, D193, K163, R192.
[074] The following mutations of l-Scel will change the preference for C in position 13 to T: Q193, C163, L163.
[075] The following mutations of l-Scel will change the preference for C in position 14 to A: L192, C192.
[076] The following mutations of l-Scel will maintain the preference for C at position 14: E161, R192, K192.
[077] The following mutations of l-Scel will change the preference for C in position 14 to G: K147, K161, R161, R197, D192, E192.
[078] The following mutations of l-Scel will change the preference for C at position 14 to T: K161, Q192.
[079] The following mutations of l-Scel will maintain the preference for C at position 15: E151.
[080] The following mutations of l-Scel will change the preference for C at position 15 to G: K151.
[081] The following mutations of l-Scel will change the preference for C in position 15 to T: C151, L151. K151.
[082] The following mutations of l-Scel will maintain the preference for A in position 17: N152, S152, C150, L150, V150, T150.
[083] The following mutations of l-Scel will change the preference for A in position 17 to C: K152, K150.
[084] The following mutations of l-Scel will change the preference for A in position 17 to G: N152, S152, D152, D150, E150.
[085] The following mutations of l-Scel will change the preference for A in position 17 to T: Q152, Q150.
[086] The following mutations of l-Scel will change the preference for
G in position 18 for A: K155, C155.
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18/71 [087] The following mutations of l-Scel will change the preference for G at position 18: R155, K155.
[088] The following mutations of l-Scel will maintain the preference for G at position 18: E155.
[089] The following mutations of l-Scel will change the preference for G at position 18 to T: H155, Y155.
[090] Combinations of several mutations can increase the effect. An example is the triple mutant W149G, D150C and N152K, which will change the preference of l-Scel for A in position 17 to G.
[091] In order to preserve enzyme activity, mutations I38S, I38N, G39D, G39R, L40Q, L42R, D44E, D44G, D44H, D44S, A45E, A45D, Y46D, I47R, I47N, D144E, D145E, D145N and G146E from l-Scel or its counterpart that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% amino acid level sequence identity should be avoided.
[092] Mutations that alter enzyme activity, DNA binding affinity, the DNA recognition sequence of l-Scel or its homologue that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acid level sequence identity can be combined to create a genetically engineered modified endonuclease, for example example, an endonuclease modified by genetic engineering based on l-Scel and which has an altered DNA binding affinity and / or an altered DNA recognition sequence when compared to l-Scel as described by SEQ ID NO: 1.
[093] In addition to the rational engineering of l-Scel, it is also possible to change the enzymatic activity, the DNA binding affinity, the DNA recognition sequence of l-Scel or its homologue that has at least 55%,
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58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity, using molecular evolution. Polynucleotides that encode a candidate endonuclease enzyme can, for example, be modulated with DNA scrambling protocols. DNA shuffling is a process of recombination and recursive mutation, performed by random fragmentation of a pool of related genes, followed by the reassembly of the fragments through a polymerase chain reaction process. See, for example, Stemmer (1994) Proc Natl Acad Sei USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; and US 5,605,793, US 5,837,458, US 5,830,721 and US 5,811,238. Genetically engineered endonucleases can also be created using rational design, based on additional knowledge of the crystalline structure of a given endonuclease, see, for example, Fajardo-Sanchez et al., Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequences, Nucleic Acids Research, 2008, Vol. 36, No. 7 2163-2173. Several examples of genetic engineering modified endonucleases, as well as their respective DNA recognition sites are known in the art and described, for example, in: WO 2005/105989, WO 2007/034262, WO 2007/047859, WO 2007/093918, WO 2008/093249, WO 2008/102198, WO 2008/152524, WO 2009/001159, WO 2009/059195, WO 2009/076292, WO 2009/114321, or WO 2009/134714, WO 10/001189, included herein by way of of reference.
[094] The mutations and alterations to create optimized nucleases can be combined with the mutations used to create genetically engineered modified endonucleases, for example, a l-Scel homolog can be an optimized nuclease as described in this document, but
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20/71 can also comprise mutations used to alter its DNA binding affinity and / or alter its DNA recognition sequence.
[095] The amino acid sequence of l-Scel or its counterparts that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% amino acid level sequence identity, as well as the polynucleotides that code for l-Scel or their counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acid level sequence identity can be improved by adapting the sequence of polynucleotide to the use of the organism's codon, where l-Scel or its counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% amino acid level sequence identity is intended to be expressed, either excluding alternative initiation codons, or excluding cryptic polyadenylation signals from the polynucleotide sequence that codes for the endonuclease.
Mutations Used to Create Optimized Nucleases:
[096] Nucleases optimized as optimized versions of l-Scel or their counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity at the amino acid level can be optimized by changing the amino acid sequence of the respective LAGLIDADG endonuclease to improve protein stability. Correspondingly, optimized nucleases do not comprise or have a reduced number compared to the amino acid sequence of the non-optimized nuclease of:
a) PEST strings,
b) KEN-boxes
c) A-boxes,
d) D-boxes, or
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e) comprise an N-terminal termination optimized for stability according to the N-terminal rule,
f) comprise a glycine as the second Nterminal amino acid, or
g) any combination of a), b), c) d), e) and f).
[097] PEST sequences are sequences of about 12 amino acids, comprising at least one proline, glutamate or aspartate and at least one serine or threonine. PEST sequences are, for example, described in Rechsteiner M, Rogers SW. “PEST sequences and regulation byproteolysis.” Trends Biochem. Sci. 1996; 21 (7), pages 267 to 271.
[098] The amino acid consensus sequence for a KEN-box is: KENXXX (N / A).
[099] The amino acid consensus sequence for an A-box is: AQRXLXXSXXXQRVL.
[0100] The amino acid consensus sequence for a D-box is: RXXL.
[0101] An additional way to stabilize the nucleases against degradation is to optimize the N-terminal amino acid sequence of the respective endonuclease according to the N-terminal rule. The nucleases that are optimized for expression in eukaryotes comprise methionine, valine, glycine, threonine, serine, alanine or cysteine after the methionine starting from its amino acid sequence. The nucleases that are optimized for expression in prokaryotes comprise methionine, valine, glycine, threonine, serine, alanine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, isoleucine or histidine after the starting methionine from its amino acid sequence. Nucleases can be further optimized by excluding 50, 40,30, 20,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids from their amino acid sequence, without destroying their endonuclease activity . For example, in a case where parts
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22/71 of the amino acid sequence of a LAGLIDADG endonuclease are excluded, it is important to retain the LAGLIDADG endonuclease motif described above.
[0102] Another way to optimize the nucleases is to add nuclear localization signals to the amino acid sequence of the nuclease. For example, a nuclear localization signal as described by SEQ ID NO: 4. The optimized nucleases can comprise a combination of the methods and resources described above, for example, they can comprise a nuclear localization signal, comprise a glycine as the second Nterminal amino acid or an exclusion in the C-terminal or a combination of these features. Examples of optimized nucleases that have a combination of the methods and resources described above are, for example, described by SEQ ID NOs: 2, 3 and 5.
[0103] The optimized nucleases do not comprise an amino acid sequence described by the sequence: HVCLLYDQWVLSPPH, LAYWFMDDGGK, KTIPNNLVENYLTPMSLAYWFMDDGGK, KPIIYIDSMSYLIFYNLIK, KLPNTISSETDK, or
KPIIYIDSMSYLIFYNLIK, KLPNTISSETFLK or TISSETFLK, or that do not comprise an amino acid sequence described by the sequence: HVCLLYDQWVLSPPH, LAYWFMDDGGK, KLPNTISSETFLK or TISSETFLK, or that do not comprise a sequence, or TK, TK, or amino acid described by the sequence: KLPNTISSETFLK or TISSETFLK.
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23/71 [0104] In one embodiment, the optimized nuclease is l-Scel, or its counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity where the TISSETFLK amino acid sequence in the wild-type IScel C-terminal or its counterparts that has at least 55% , 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity and which has a TISSETFLK amino acid sequence at the C-terminus is deleted or mutated.
[0105] The amino acid sequence TISSETFLK can be deleted or mutated, excluding or mutating at least 1,2,3, 4, 5, 6, 7, 8 or 9 amino acids from the C-terminal of l-Scel type wild or its counterparts that owns at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid level sequence identity and that has a TISSETFLK amino acid sequence in the Cterminal.
Table 2: Different examples for exclusions of the amino acid sequence
TISSETFLK in I- Scel wild type:
l-Scel wild type and optimized C-terminal amino acid sequence l-Scel wild type TISSETFLK l-Scel -1 TISSETFL l-Scel -2 TISSETF l-Scel -3 TISSET l-Scel -4 TISSE l-Scel -5 TISS l-Scel -6 TIS l-Scel -7 YOU l-Scel -8 T l-Scel -9 Complete exclusion
[0106] In an embodiment of the invention, the optimized nucleases or optimized versions of l-Scel and their counterparts that have at least 55%,
58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% amino acid level sequence identity comprises at least
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24/71 minus one of the following mutations: L74K, Y75H, Q77K, E130K, T134H, Y199H, M203K, Y205H.
[0107] Likewise, it is preferred to mutate the serine at position 229 of the wild-type l-Scel amino acid sequence as described in SEQ ID NO: 1 for Lys, Ala, Pro, Gly, Glu, Gin, Asp, Asn, Cys , Tyr or Thr. Thus creating the mutants l-Scel S229K, S229A, S229P, S229G, S229E, S229Q, S229D, S229N, S229C, S229Y, or S229T. Amino acid No. 229 of wild-type l-Scel is amino acid Nr. 230 in SEQ ID NO: 2.
[0108] In another embodiment of the invention, the amino acid methionine at position 202 of the wild-type l-Scel amino acid sequence as described in SEQ ID No. 1 (amino acid 203 if referenced to SEQ ID No. 2), is mutated to Lys, His or Arg. Thus creating the mutant l-Scel M202K, M202H and M202R.
[0109] Alternatively, the amino acid sequence TISSETFLK can be mutated, for example, to the amino acid sequence: TIKSETFLK, or AIANQAFLK.
[0110] The preferred optimized versions of l-Scel are the exclusions l-Scel -1, l-Scel -2, l-Scel -3, l-Scel - 4, l-Scel -5, l-Scel -6, l-Scel -7,1Scel -8, l-Scel -9 and the mutants S229K and S229A, more preferably, are the exclusions l-Scel -1, l-Scel -2, l-Scel -3, l-Scel -4, l-Scel -5, l-Scel -6 and the mutant S229K. Most preferred are the exclusion of l-Scel -5 (SEQ ID O 30) and the mutant S229K.
[0111] It is also possible to combine the exclusions and mutations described above, for example, by combining the exclusion of l-Scel -1 with the mutant S229A, thus creating the amino acid sequence TIASETFL at the C-terminal.
[0112] Optimized versions plus l-Scel preferences are the l-Scel -1, l-Scel -2, l-Scel -3, 1- Scel -4, l-Scel -5, l-Scel -6 exclusions, l-Scel -7.1
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Scel -8, l-Scel -9 or the S229K and S229A mutants, in combination with the M202K mutation.
[0113] Even more preferred are the exclusions l-Scel -1, IScel -2, l-Scel -3, l-Scel -4, l-Scel -5, l-Scel - 6 or the mutant S229K in combination with the M202K mutation. In another embodiment of the invention, the amino acids glutamine at position 76, glutamic acid at position 129, or tyrosine at position 198 of the wild-type l-Scel amino acid sequence as described in SEQ ID No. 1 (amino acids 77, 130 and 199 being if referenced to SEQ ID No. 2), they are mutated to Lys, His or Arg. Thus creating the mutants l-Scel Q76K, Q76H, Q76R, E129K, E129H, E129R, Y198K, Y198HeY198R.
[0114] The exclusions and mutations described above will also apply to their l-Scel counterparts that have at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity at the amino acid level and which has a TISSETFLK amino acid sequence at the C-terminus.
[0115] Consequently, in an embodiment of the invention, the optimized endonuclease is an optimized version of l-Scel or one of its counterparts that has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acid level sequence identity, and which has one or more mutations or exclusions selected from the group of : l-Scel -1, l-Scel -2, I-Scel -3, l-Scel -4, l-Scel -5, l-Scel -6,1Scel -7, l-Scel -8, l-Scel -9, S229K, S229A, S229P, S229G, S229E, S229Q, S229D, S229N, S229C, S229Y, S229T, M202K, M202H, M202R, Q76K, Q76H, Q76R, E129K, E129H, E129H, E9 that the amino acid numbers are referenced to the amino acid sequence as described by SEQ ID NO: 1. In an additional embodiment of the invention, the optimized endonuclease is an optimized version of l-Scel or one of its counterparts that
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26/71 has at least 55%, 58%, 60%, 70%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity at the amino acid level, and which has one or more mutations or exclusions selected from the group of: l-Scel -1, l-Scel -2, l-Scel -3, l-Scel -4, l -Scel -5, l-Scel -6, S229K and M202K, where the amino acid numbers are referenced to the amino acid sequence as described by SEQ ID NO: 1.
[0116] A particular preferred optimized endonuclease is a wild type or genetically engineered version of l-Scel, as described by SEQ ID NO: 1 or one of its counterparts that has at least 55%, 58%, 60%, 70 %, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of amino acid level sequence identity and which has one or more mutations selected from groups of:
a) l-Scel -1, l-Scel -2, l-Scel -3, l-Scel -4, l-Scel -5, l-Scel -6, IScel -7, l-Scel -8 and l- Scel -9;
b) S229K, S229A, S229P, S229G, S229E, S229Q, S229D, S229N, S229C, S229Y, S229T, M203K, M203H, M203R, Q77K, Q77H, Q77R, E 130K, E130H, E1309, 19
c) methionine, valine, glycine, threonine, serine, alanine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, isoleucine or histidine after the methionine starting from its amino acid sequence; or
d) a combination of one or more mutations selected from a) and b), a) and c), b) and c) or a) b) and c) above.
[0117] The optimized endonuclease is preferably expressed as a fusion protein with a nuclear localization sequence (NLS). This NLS sequence allows easier transport to the nucleus and increases the efficiency of the recombination system. A variety of NLS sequences are known to well-versed individuals and described, among others, by Jicks G and Raikhel NV (1995) Annu. Rev. Cell Biol. 11: 155-188. For example, the sequence
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Large SV40 antigen NLS is preferred for plant organisms. Examples are provided in WO 03/060133. NLS can be heterologous to the endonuclease and / or the DNA binding domain or can be naturally understood in the endonuclease and / or the DNA binding domain.
[0118] Another embodiment of the invention consists of translational fusions comprising optimized endonucleases and heterologous DNA binding domains. Optimized endonucleases comprise mutations as described above and may or may not comprise additional mutations as described above, for example, the mutations used to create engineered endonucleases.
[0119] Preferred heterologous DNA binding domains are zinc finger (fingert zinc or repeat units derived from a transcription activator-type effector (TAL) (also known as TAL repeat).
[0120] Consequently, in one embodiment of the invention, the optimized endonuclease is fused to at least one zinc finger domain (zinc fingeh, or at least one of the repeat units derived from a transcription-activating effector (TAL ), or at least one zinc finger domain (zinc fingef and at least one of the repeat units derived from a transcription activator-type effector (TAL).
[0121] Those mergers can be N-terminal or C-terminal or N- and C-terminal to the optimized endonulease.
[0122] For example, it is possible to fuse at least one zinc finger domain (zinc fingerj to the N-terminal and at least one zinc finger domain (zinc finger) to the C-terminal of the optimized endonuclease, or to fuse at least one domain of zinc finger (zinc finger) to the N-terminal and at least one repeating unit derived from an effector type activator of
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28/71 transcription (TAL) to the C-terminal of the optimized endonuclease. Alternatively, it is also possible to fuse a combination of at least one zinc finger domain and at least one repeat unit derived from a transcription activator-type effector (TAL) to the N- or C-terminal or to the N- and C-terminal of an optimized endonuclease. Basically any permutation of these elements is possible. The zinc finger domains have conserved cysteine and histidine residues that tetrahedrally coordinate the single zinc atom in each finger domain. In particular, most ZFPs are characterized by finger components of the general sequence:
-Cys- (X) 2-4-Cys- (X) i2-His- (X) 3-5-His-, where X represents any amino acid (the C2H2 ZFPs). The zinc finger domains of this most widely represented class contain two cysteines and two histidines with particular spacing. The folded structure of each data domain contains an antiparallel beetle, a finger tip region and a short antipathic alpha 10 helix. The coordinating metal ligands bind to the zinc ion and, in the case of a zif268-type zinc finger, the short antipathetic a-helix binds to the main DNA slot. In addition, the structure of the zinc fingers is stabilized by certain conserved hydrophobic amino acid residues (for example, the residue directly before the first conserved Cys and the residue at the +4 position of the helical segment of the finger) and by coordination of zinc 15 through conserved cysteine and histidine residues. Canonical C2H2 ZFPs have been described that have changes in the positions that make direct base contacts, 'support' or 'support' residues immediately adjacent to the base contact positions, and positions capable of coming into contact with the DNA phosphate backbone. . See, for example, U.S. Patent Nos. 6,007,988; 6,013,453; 6,140,081; 6,866,997; 6,746,838; 6,140,081; 6,610,512; 7,101,972; 6,453,242; 6,785,613; 7,013,219; PCT WO
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98/53059; Choo et al. (2000) Curro Opin. Struct. Biol.10: 411-416; Segai et al. (2000) Curro Opin. Chern. Biol.4: 34-39.
[0123] Furthermore, zinc finger proteins (zinc pretend / conque has zinc fingers with modified zinc coordination residues have also been described (see, for example, US Patent Applications Nos. 25 20030108880, 20060246567 and 20060246588 ; the descriptions of which are hereby incorporated by reference).
[0124] The terms "repeat unit derived from a transcription activator (TAL) effector", "repeat unit" and "TAL repeat" are used interchangeably and are used to describe the modular portion of a domain of repetition from a TAL effector, or an artificial version thereof, which contains two amino acids at positions 12 and 13 of the amino acid sequence of a repeat unit that determines the recognition gave a base pair in a target DNA sequence of such so that amino acids recognize: HD for C / G recognition; NI for AT recognition; NG for T / A recognition; NS for recognition of C / G or AT or T / A or G / C; NN for G / C or A / T recognition; IG for T / A recognition; N for C / G recognition; HG for C / G or T / A recognition; H for T / A recognition; and NK for G / C recognition, (amino acids H, D, I, G, S, K are described by one-letter codes, so A, T, C, G refer to the recognized DNA base pairs by amino acids).
[0125] The number of repetition units to be used in a repetition domain can be ascertained by an individual skilled in the technique through routine experimentation. In general, at least 1.5 repetition units are considered to be a minimum, although typically at least about 8 repetition units are used. Repeating units do not
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30/71 must be complete repetition units, since half size repeating units can be used. A hetetological DNA-binding domain of the invention can comprise, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 , 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15 , 5, 16, 16,5, 17, 17,5, 18, 18,5, 19, 19,5, 20, 20,5, 21,21,5, 22,
22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5,
31.31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39,
39.5, 40, 40.5, 41.41.5, 42, 42.5, 43, 43.5, 44, 44.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5 or more repetition units.
[0126] A typical consensus sequence for a 34 amino acid repeat (in one letter code) is shown below:
LTPEQWAIASNGGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 19).
[0127] An additional consensus sequence for a 35 amino acid repeat unit (in one letter code) is as follows: LTPEQWAIASNGGGKQALETVQRLLPVLCQAPHD (SEQ ID NO: 20).
[0128] The repeating units that can be used in an embodiment of the invention have an identity with the consensus sequences described above of at least 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.
[0129] Zinc finger domains (zinc fingeh, as well as TAL repeats can be mutated to bind to any given polynucleotide sequence. Methods on how to select the appropriate mutations are described in W00027878, WO03062455, W008076290, W008076290, W09945132 and WO2010 / 079430, which are included here for reference, so it is possible to select a polynucleotide sequence next to an optimized endonuclease DNA recognition sequence, and to mutate the zinc finger domains or the repeats TAL to bind to that neighboring polynucleotide sequence.
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31/71 zinc finger or TAL repeats can then be used for translational fusions with the respective optimized endonuclease, which has the next DNA recognition sequence.
[0130] It is also possible to choose a polynucleotide sequence similar to an DNA recognition sequence of an optimized endonuclease, however, being recognized ineffectively and / or cut by the optimized endonuclease. It is possible to create translational fusions of optimized endonucleases with at least one zinc finger or TAL repeat, link to a polynucleotide sequence close to this non-optimal DNA recognition site, which will recognize and cut said recognition site of non-optimal DNA more efficiently.
[0131] It is possible to generate optimized LAGLIDADG nuclease fusions with a combination of TAL repeat and zinc finger domains. Since TAL effectors are able to recognize regions rich in AT, this can compensate for the limitation of zinc finger domains, which preferentially bind to regions rich in GC.
[0132] The TAL repeat and zinc finger domains can be used to create N-terminal or C-terminal or N-terminal and Cterminal fusions to optimized LAGLIDADG nucleases, with several TAL repetitions and / or domains zinc finger, as well as combinations thereof, can be fused at the N-terminal or C-terminal end of the optimized LAGLIDADG nucleases.
[0133] The exemplary structures of these mergers are:
N-term-1-Scel- repeat TAL (x) -C-term;
N-term- repetition TAL (x) 1-Scel-C-term;
N-term- TAL repetition (x) l-Scel- TAL-C-term repetition;
N-term-l-Scel- zinc finger domain (x) -C-term;
N-term- zinc finger domain (x) l-Scel-C-term;
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N-term- zinc finger domain (x) l-Scel- zinc finger domain (x) -C-term;
N-term- repeat TAL (x) -l-Scel- zinc finger domain (zinc finger) (x) -C-term;
N-term- zinc finger domain (x) l-Scel- TAL-C-term repeat;
N-term- repeat TAL (x) -l-Scel- zinc finger domain (zinc finger) (x) -C-term;
N-term- zinc finger domain (x) l-Scel- TAL-C-term repeat;
N-term- zinc finger domain (zinc finger) (x) -TAL repeat (x) -l-Scel- zinc finger domain (zinc finger) (x) -C-term;
N-term- zinc finger domain (zinc finger) (x) l-Scel- TAL repetition (x) -zinc finger domain (zinc finger) (x) -C-term, where (x) means a or several TAL repetitions or zinc finger domains.
[0134] In a preferred embodiment, the sequences encoding the optimized endonucleases are modified by inserting an intron sequence. This prevents the expression of a functional enzyme in prokaryotic host organisms and thus facilitates cloning and transformation procedures (for example, based on E.coli or Agrobacterium). In eukaryotic organisms, for example, plant organisms, the expression of a functional enzyme is performed, since the plants are able to recognize the introns and "splicing" them. Preferably, introns are inserted into the optimized endonucleases mentioned as preferred above.
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33/71 [0135] In another preferred embodiment, the amino acid sequences of the optimized endonuclease can be modified by adding a Sec IV secretion signal to the N-, or C-terminal of the optimized endonuclease.
[0136] In a preferred embodiment, the SeclV secretion signal is a SeclV secretion signal comprised of Vir de Agrobacteríum proteins. Examples of these Sec IV secretion signals, as well as methods of how to apply these are described in WO 01 89283, in Vergunst et al. The positive charge is an important resource of the C-terminal transport signal of the VirB / D4 translocated proteins from Agrobacteríum , PNAS 2005, 102, 03, pages 832 to 837.
[0137] A Sec IV secretion signal can also be added, by adding fragments of a Vir protein or even a complete Vir protein, for example, a complete VirE2 protein to an optimized endonuclease, in a similar manner as described in the description of WO01 / 38504, which describes a RecA / VirE2 fusion protein.
[0138] In another preferred embodiment, the amino acid sequences of the optimized endonuclease can be modified by adding a Sec III secretion signal to the N-, or C-terminal of the optimized endonuclease. Suitable Seclll secretion signals are, for example, described in WO 00/02996.
[0139] In case a Sec III secretion signal is added, it may be advantageous to express the optimized endonuclease in a cell, which also comprises a recombinant construction that encodes parts or an entire type III functional secretion system, for the purpose of super -express or complement parts or an entire type III functional secretion system in such a cell.
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34/71 [0140] Recombinant constructs encoding parts or an entire type III functional secretion system are, for example, described in WO 00/02996.
[0141] If a SeclV secretion signal is added to the optimized endonuclease and if the optimized endonuclease is to be expressed, for example, in Agrobacterium rhizogenes or Agrobacterium tumefaciens, it is advantageous to adapt the DNA sequence encoding the optimized endonuclease to codon use of the expression organism. Preferably, the optimized endonuclease lacks, or has only a few DNA recognition sequences in the genome of the expression organism. This is even more advantageous, if the optimized endonuclease does not have a DNA recognition sequence or a less preferred DNA recognition sequence in the Agrobacterium genome. If the optimized endonuclease is to be expressed in a prokaryotic organism, the sequence encoding the optimized endonuclease must not have an intron.
Polynucleotides:
[0142] The invention also comprises isolated polynucleotides that encode the optimized endonucleases described above.
[0143] Examples of such isolated polynucleotides are isolated polynucleotides that encode amino acid sequences described by SEQ ID NO: 3, 5, or amino acid sequences that have at least 70%, 80%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence similarity, preferably having at least 70%, 80%, 90% 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any of the amino acid sequences described by SEQ ID NO: 2, 3, 5.
[0144] Preferably, the isolated polynucleotide has a codon use optimized for expression in a particular host organism, or
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35/71 has a low content of NA instability grounds, or has a low content of codon repetitions, or has a low content of cryptic splicing sites, or has a low content of alternative starting codons, or has a low content of restriction sites, either have a low content of secondary RNA structures or have any combination of these resources.
[0145] The codon use of the isolated polypeptide can be optimized, for example, for expression in plants, preferably in a vegetable selected from the group comprising: rice, corn, wheat, canoe, sugar cane, sunflower , beet, potato or tobacco.
[0146] Preferably, the isolated polynucleotide is combined with a promoter sequence and as a suitable terminator sequence to form a functional expression cassette for expression of the optimized endonuclease in a particular host organism.
[0147] Suitable promoters are, for example, specific constitutive promoters, heat-inducible or pathogens of seeds, pollen, flowers or fruits.
[0148] Individuals skilled in the art know several promoters who have these resources.
[0149] For example, several plant-building promoters are known. Most of them derive from viral or bacterial sources, such as the nopaline (nos) promoter (Shawetal. (1984) NucleicAcids Res. 12 (20): 7831 -7846), the mannopine synthase promoter (mas) (Cornai et al. (1990 ) PlantMolBiol 15 (3): 373-381), or the octopine synthase (ocs) promoter (Leisner and Gelvin (1988) Proc NatlAcad Sei USA 85 (5): 2553-2557) from Agrobacterium tumefaciens or the CaMV35S promoter of Cauliflower Mosaic (US 5,352,605). The latter was most often used in the constitutive expression of transgenes in plants (Odell et al. (1985) Nature 313: 810-812; Battraw and Hall (1990) Plant Mol Biol 15: 527-538; Benfey et al. (1990) EMBO J 9 (69): 1677-1684; US 5,612,472). However, the promoter CaMV 35S
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36/71 demonstrates variability not only in different plant species, but also in different plant tissues (Atanassova et al. (1998) Plant Mol Biol 37: 275-85; Battraw and Hall (1990) Plant Mol Biol 15: 527-538; Holtorf et al. (1995) Plant Mol Biol 29: 637-646; Jefferson et al. (1987) EMBO J 6: 3901-3907). An additional disadvantage is an interference of the transcriptional regulatory activity of the 35S promoter with the wild-type CaMV virus (Al-Kaff et al. (2000) Nature Biotechnology 18: 99599). Another viral promoter for constitutive expression is the Sugarcane bacilliform badnavirus (ScBV) promoter (Schenketal. (1999) PlantMolBiol39 (6): 1221-1230).
[0150] Various plant constitutive promoters are described, such as the Arabidopsis thaliana ubiquitin promoter (Callis et al. (1990) J Biol Chem 265: 12486-12493; Holtorf S et al. (1995) Plant Mol Biol29-.637 -747), which - however - is reported to be unable to regulate the expression of selection markers (W003102198), or two corn ubiquitin promoters (Ubi-1 and U bi-2; US 5,510,474; US 6,020 .190; US 6,054,574), which in addition to a constitutive expression profile demonstrates an induction of thermal shock (Christensen et al. (1992) Plant. Mol. Biol. 18 (4): 675-689). A comparison of the level of specificity and expression of CaMV 35S, the barley thionine promoter, and the Arabidopsis ubiquitin promoter based on stably transformed Arabidopsis plants demonstrate a high rate of expression for the CaMV 35S promoter, while the promoter of Thionine was inactive in most strains and the Arabisopsis promoter resulted in only moderate expression activity (Holtorf et al. (1995) Plant Mol Biol29 (4): 637-6469).
Vectors:
[0151] The polynucleotides described above can be comprised in a DNA vector suitable for transformation, transfection, cloning or overexpression.
[0152] In one example, the polynucleotides described above are comprised of a vector for transforming
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37/71 non-human organisms or cells, preferably non-human organisms are plants or plant cells.
[0153] In general, the vectors of the invention comprise additional functional elements, which may include, but are not limited to:
i) Origins of replication that guarantee the replication of the expression cassettes or vectors according to the invention, for example, in E. coli. Examples that can be mentioned are ORI (origin of DNA replication), pBR322 ori or ο P15A ori (Sam-brook et al .: Molecular Cloning. A Laboratory Manual, 2- ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, 1989);
ii) Multiple cloning sites (MCS) to allow and facilitate the insertion of one or more nucleic acid sequences;
iii) Sequences that make possible a homologous recombination or insertion in the genome of a host organism;
iv) Elements, for example, bounding sequences, that make transference mediated by Agrobacterium in plant cells possible for transfer and integration into the plant genome, such as, for example, the right or left boundary of T-DNA or the vir region .
The Marker Sequence [0154] It should be understood, in the broad sense, that the term "marker sequence" includes all nucleotide sequences (and / or polypeptide sequences translated from them) that facilitate the detection, identification, or selection of transformed cells, tissues or organisms (eg plants). The terms "sequence that allows the selection of a transformed plant material", "selection marker" or "selection marker gene" or "selection marker protein" or "marker" have essentially the same meaning.
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38/71 [0155] Markers can include (but are not limited to) a selectable marker and a traceable marker. A selectable marker gives the cell or organism a phenotype that results in a difference in growth or viability. The selectable marker can interact with a selection agent (such as an herbicide or antibiotic or prodrug) to produce this phenotype. A screenable marker gives the cell or organism a readily detectable phenotype, preferably a visibly detectable phenotype such as a color or color. The screenable marker can interact with a screening agent (such as a dye) to produce this phenotype.
[0156] The selectable marker (or selectable marker strings) comprises, but is not limited to
a) negative selection marker, which confers resistance against one or more toxic agents (in the case of phytotoxic plants) such as antibiotics, herbicides or other biocides,
b) counter-selection marker, which confers a sensitivity against certain chemical compounds (for example, converting a non-toxic compound to a toxic compound), and
c) positive selection marker, which gives a growth advantage (for example, through expression of key elements of the cytokine or hormone biosynthesis that leads to the production of a plant hormone, for example, auxin, guibellins, cytokines, abscisic acid and ethylene; Ebinuma H et al. (2000) Proc Natl Acad Sei USA 94:21 17-2121).
[0157] When using negative selection markers, only cells or plants are selected comprising said negative selection marker. When using a counter-selection marker, only cells or plants that are devoid of said counter-selection marker are selected. The counter-selection marker can be used to check the excision
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39/71 successful sequence (comprising said counter-selection marker) from a genome. Screenable marker sequences include, but are not limited to, reporter genes (for example, luciferase, glucuronidase, chloramphenicol acetyl transferase (CAT, etc.). Preferred marker sequences include, but are not limited to:
i) Negative selection marker [0158] Normally, negative selection markers are useful for selecting cells that have been successfully subjected to transformation. The negative selection marker, which was introduced with the DNA construct of the invention, can confer resistance to a biocide or phytotoxic agent (for example, a herbicide such as phosphinothricin, glyphosate or bromoxynil), a metabolism inhibitor such as 2-deoxyglucose-6 -phosphate (WO 98/45456) or an antibiotic such as, for example, tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin or hygromycin to cells that have been successfully subjected to transformation. The negative selection marker allows selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81 -84). The negative selection marker in a vector of the invention can be used to confer resistance in more than one organism. For example, a vector of the invention may comprise a selection marker for amplification in bacteria (such as E.coli or Agrobacterium) and plants. Examples of selectable markers for E. coli include: genes that specify antibiotic resistance, that is, ampicillin, tetracycline, kanamycin, erythromycin, or genes that confer other types of selectable enzyme activities such as galactosidase, or the lactose operon. Selectable markers suitable for use in mammalian cells include, for example, the dihydrofolate reductase (DHFR) gene, the thymidine kinase (TK) gene, or prokaryotic genes that confer drug resistance, gpt (xanthine-guaninephosphorphosyltransferase, which can be selected
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40/71 with mycophenolic acid; neo (neomycin phosphotransferase), which can be selected with G418, hygromycin, or puromycin; and DHFR (dihydrofolate reductase), which can be selected with methotrexate (Mulligan & Berg (1981) Proc NatlAcad Sei USA 78: 2072; Southern & Berg (1982) J Mol Appl Genet 1: 327). Generally, selection markers for plant cells confer resistance to a biocide or antibiotic, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, or resistance to herbicide, such as resistance to chlorsulfurone or Basta.
[0159] Especially preferred negative selection markers are those that confer resistance to herbicides. Examples of negative selection markers are:
the DNA sequences encoding phosphinothricin acetyltransferases (PAT), which acetylates the free amino group of the inhibitor glutamine phosphinothricin synthase (PPT) and therefore produces PPT detoxification (from Block et al. (1987) EMBO J 6: 2513- 2518) (also referred to as Bialophos resistant bar gene; EP 242236), 5-enolpyruvylshikimato-3-phosphate synthase genes (EPSP synthase genes), which confer resistance to Glyphosate- (N- (phosphonomethyl) glycine), the gox gene, which encodes the glyphosate degradation enzyme oxydoreduetase, the deh gene (which encodes a dealogenase that inactivates Dalapon-), acetolactate synthases that confer resistance to sulfonylurea and imidazolinone, bxn genes that encode bromoxinil degradation nitrilase enzymes, kanamycin , or G418, resistance gene (NPTII). The NPTII gene encodes a neomycin phosphotransferase that reduces the inhibitory effect of
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41/71 kanamycin, neomycin, G418 and paromomycin due to a phosphorylation reaction (Becket al (1982) Gene 19: 327), the DOGR1 gene. The DOGR1 gene was isolated from the yeast Saccharomyces cerevisiae (EP 0 807 836). This encodes a 2-deoxyglucose-6 phosphate phosphatase that confers resistance to 2-DOG (Randez-Gil et al. (1995) Yeasí11: 1233-1240).
the hyg gene, which encodes the enzyme hygromycin phosphotransferase and confers resistance to the antibiotic hygromycin (Gritz and Davies (1983) Gene 25: 179);
Especially preferred are the negative selection markers that confer resistance against the toxic effects imposed by type D amino acids, for example, D-alanine and D-serine (WO 03/060133; Erikson 2004). Especially preferred as a negative selection marker in this context are daol genes (EC: 1.4. 3.3: GenBank Acc.-No .: U60066) from the yeast Rhodotorula gracilis (Rhodosporidium toruloides) and E. coli dsdA gene (D-serine dehydratase ( Dserine deaminase) (EC: 4.3.1.18; GenBank Acc.-No .: J01603).
ii) Positive selection marker [0160] The positive selection marker comprises, but is not limited to, growth stimulus selection marker genes such as Agrobacteríum tumefaciens isopentenyl transferase (strain: P022; Genbank Acc.-No .: AB025109) it can-as a key enzyme of cytokine biosynthesis - facilitate the regeneration of transformed plants (for example, by selection in a cytokine-free medium). The corresponding selection methods are described (Ebinuma H et al. (2000) Proc Natl Acad Sei USA 94:21 17-2121; Ebinuma H et al. (2000) Selection of Marker-free transgenic plants using the oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable markers, Molecular Biology of Woody Plants, Kluwer Academic Publishers). The additional positive selection markers, which give a growth advantage to a
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42/71 transformed vegetable compared to untransformed, are described, for example, in EP-A 0 601 092. Growth stimulus selection markers may include (but are not limited to) beta-glucuronidase (in combination with, for example, a cytokinin glucuronide), mannose-6-phosphate isomerase (in combination with mannose), UDP-galactose-4-epimerase (in combination with, for example, galactose), with mannose-6-phosphate isomerase in combination with especially preferred mannose PE.
ui) Counter-selection markers [0161] The counter-selection marker allows the selection of organisms with sequences successfully excluded (KoprekT et al. (1999) Plant J 19 (6): 719-726). Fragment of TKtimidine kinase (TK) and diphtheria toxin A (DTA), codA gene encoding a cytosine deaminase (Gleve AP et al. (1999) Plant Mol Biol 40 (2): 223-35; Pereat RI et al. (1993 ) Plant Mol Biol 23 (4) 793-799; Stougaard J (1993) Plant J 3: 755-761), the cytochrome P450 gene (Koprek et al. (1999) Plant J 16: 719- 726), genes encoding a haloalkane dealogenase (Naested H (1999) Plant J 18: 571 -576), the iaaH gene (Sundaresan V et al. (1995) Genes & Development 9: 1797-1810), the tms2 gene (Fedoroff NV & Smith DL ( 1993) Plant J 3: 273-289), and the D-amino oxidases which cause toxic effects by converting D-amino acids (WO 03/060133).
[0162] In a preferred embodiment, the excision cassette includes at least one of said counter-selection markers to distinguish plant cells or plants with sequences successfully excised from the plant that still contains these. In a more preferred embodiment, the excision cassette of the invention comprises a double function marker, that is, a marker that can be used as a negative selection marker or as a counter-selection marker depending on the substrate used in the selection scheme. . An example for a double-function marker is the yeast daol gene (EC: 1.4. 3.3: GenBank Acc.-No .: U60066)
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Rhodotorula gracilis, which can be used as a negative selection marker with D-amino acids such as D-alanine and D-serine, and as a counter-selection marker with D-amino acids such as D-isoleucine and D-valine (see Order for European Patent No .: 04006358.8)
IV) SCREENABLE MARKER (REPORTER GENES) [0163] The screeningable marker (such as reporter genes) encodes readily quantifiable or detectable proteins and which, through an intrinsic color or enzymatic activity, guarantees the evaluation of the transformation efficacy or the expression location or timing. Especially preferred are the genes that encode reporter proteins (see also Schenborn E, Groskreutz D. (1999) MolBiotechnol 13 (1): 29-44) as “green fluorescent protein” (GFP) (Chui WL et al. (1996) Curr Biol6: 325-330; Lef-fel SM et al. (1997) Biotechniques23 (5): 912-8; Sheen et al. (1995) Plant J 8 (5): 777-784; Haseloff et al. (1997 ) Proc Natl Acad Sei USA 94 (6): 2122-2127; Reichel et al. (1996) Proc Natl Acad Sei USA 93 (12): 5888-5893; Tian et al. (1997) Plant Cell Rep 16: 267- 271; WO 97/41228);
- Chloramphenicol transferase,
- luciferase (Millar et al. (1992) Plant Mol Biol Rep 1 0: 324-414; Ow et al. (1986) Science 234: 856-859) allows selection by detection of bioluminescence,
- beta-galactosidase, encodes an enzyme for which a variety of chromogenic substrates are available,
- beta-glucuronidase (GUS) (Jefferson et al. (1987) EMBO J 6: 3901-3907) or the uidA gene, which encodes an enzyme for a variety of chromogenic substrates,
- R locus gene product: protein that regulates the production of anthocyanin pigments (red coloring) in plant tissue and, therefore, makes possible the direct analysis of the promoter activity without the addition of
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44/71 adjuvants or additional chromogenic substrates (Dellaporta et al. (1988) In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 1 1: 263-282,),
- beta-lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA 75: 3737-3741), enzyme for a variety of chromogenic substrates (eg PADAC, a chromogenic cephalosporin),
- xylE gene product (Zukowsky et al. (1983) Proc Natl Acad Sci USA 80: 1 101-1 105), catechol dioxigenase capable of converting chromogenic catechols,
- alpha-amylase (Ikuta et al. (1990) Bio / technol. 8: 241-242),
- tyrosinase (Katz et al. (1983) J Gene Microbiol 129: 2703-2714), an enzyme that oxidizes tyrosine to provide DOPA and dopaquinone that subsequently forms melanin, which is readily detectable,
-aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126 (3): 1259-1268), can be used in the detection of calcium-sensitive bioluminescence.
Target Organisms [0164] Any organism suitable for transformation or distribution of an optimized endonuclease can be used as a target organism. This includes prokaryotes, eukaryotes, and archaea, in particular, human or animal cells, animals, plants, fungi or yeasts, preferably plants, fungi or yeasts.
[0165] In one embodiment, the target organism is a vegetable.
[0166] The term "plant" includes complete plants, vegetative organs / structures of branches (eg leaves, stems and bulbs), roots, flowers and floral organs / structures (eg bracts, sepals, petals, stamens, carpels , anthers and ova), seeds (including embryos, endosperm, and integuments) and fruits (the mature ovary), plant tissues (for example, tissue
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45/71 vascular, primary tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like), and offspring thereof. In general, the class of plants that can be used in the method of the invention is as broad as the class of upper and lower plants receptive to transformation techniques, including angiosperms (monocot and dicot plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
[0167] All genera and species of upper and lower plants of the plant kingdom are included in the scope of the invention. In addition, mature plants, seeds, branches and seedlings, and parts, propagating material (for example, seeds and fruits) and cultures, for example, cell cultures, derived from them are included.
[0168] Plants and plant materials of the following plant families are preferred: Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, osaceae, Saxifragaceae, Saxifragaceae, Scrifragaceae, Saxifragaceae, Scrifragaceae, Scrifragaceae, Scrifragaceae, Saxifragaceae, Saxifragaceae, Saxifragaceae, Scrifragaceae, Saxifragaceae, Scrifragaceae, Saxifragaceae, Scrifragaceae, Saxifriacaceae, Scrifragaceae, Tetra.
[0169] Annual perennial monocot and dicot plants are the preferred host organisms for the generation of transgenic plants. The use of the recombination system, or method according to the invention, is additionally advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or peat. The said vegetable can include - but is not limited to - bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); pteridophytes such as ferns, ponytails and Lycopodiophyta; gymnosperms such as conifers, cicadea, ginkgo and Gnetaeae; algae like Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.
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46/71 [0170] Plants for the purposes of the invention may comprise Rosaceae families as roses, Ericaceae as rhododendrons and azaleas, Euphorbiaceae as calla lily and croton, Caryophyllaceae as carnations, Solanaceae as petunias, Gesneriaceae as African violet , Balsaminaceae as balsamine, Orchidaceae as orchids, Iridaceae as gladioli, iris, freesia and saffron, Compositae as marigold, Geraniaceae as geranium, Liliaceae as dracaena, Moraceae as ficus, Araceae as philodendron and many others.
[0171] The transgenic plants according to the invention are additionally selected in particular from dicotyledonous crop plants such as, for example, from the Leguminosae families such as peas, alfalfa and soybeans; Solanaceae as tobacco and many others; the Umbelliferae family, particularly the genus Daucus (particularly the species carota (carrot)) and Apium (particularly the species graveolens dulce (celery)) and many others; the Solanaceae family, particularly the genus Lycopersicon, particularly the species esculentum (tomato) and the genus Solanum, particularly the species tuberosum (potato) and melongena (eggplant) and many others; and the genus Capsicum, particularly the species annum (pepper) and many others; the Leguminosae family, particularly the Glycine genus, particularly the max (soy) specimen and many others; and the family of Cruciferae, particularly the genus Brassica, particularly the species napus (rapeseed), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleraceacv Emperor (broccoli); and the genus Arabidopsis, particularly the thaliana species and many others; the Compositae family, particularly the Lactuca genus, particularly the sativa species (lettuce) and many others.
[0172] The transgenic plants according to the invention are selected in particular from monocot crop plants,
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47/71, such as cereals such as wheat, barley, sorghum and millet, rye, triticalo, maize, rice or oats, and sugar cane. Especially preferred are Arabidopsis thaliana, Nicotiana tabacum, rapeseed, soybeans, maize (maize), wheat, flax seed, potatoes and tagetes.
[0173] Additionally, plant organisms, for the purpose of the invention, are other organisms that are capable of photosynthetic activity, such as, for example, algae or cyanobacteria, and also mosses. The preferred algae are green algae, for example, algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.
[0174] Plants genetically modified according to the invention that can be consumed by humans or animals can also be used as food or animal feed, for example, directly or following the processing known in the art.
Construction of Polynucleotide Constructions [0175] Typically, polynucleotide constructions (for example, for an expression cassette) to be introduced into a non-human organism or cells, for example, plants or plant cells are prepared using transgenic expression. Recombinant expression techniques involve the construction of recombinant nucleic acids and the expression of genes in transfected cells. Molecular cloning techniques to achieve these purposes are known in the art. A wide variety of in vitro cloning and amplification methods suitable for the construction of recombinant nucleic acids are well known to those skilled in the art. Examples of these techniques and sufficient instructions to instruct individuals versed in the technique through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, hie, San Diego, CA (Berger ); Current Protocols in Molecular Biology, F.
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M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement), T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning : A Laboratory Manua , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984). Preferably, the DNA constructs employed in the invention are generated by joining the aforementioned essential constituents of the DNA construction together in the aforementioned sequence using the recombination and cloning techniques with which those skilled in the art are familiar.
[0176] The construction of polynucleotide constructs generally requires the use of vectors capable of replicating in bacteria. A plethora of kits is commercially available for purifying plasmids from bacteria. The isolated and purified plasmids can then be further manipulated to produce other plasmids, used to transfect cells or incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes to infect and transform plants. When Agrobacterium is the means of transformation, the shuffling vectors are constructed.
Methods for Introducing Constructions in Target Cells [0177] A DNA construct employed in the invention can advantageously be introduced into cells using vectors where said DNA construct is inserted. Examples of vectors can be plasmids, cosmids, phages, viruses, retroviruses or agrobacteria. In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. The preferred vectors are those that allow the stable integration of the expression cassette into the host genome.
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49/71 [0178] A DNA construct can be introduced into target plant cells and / or organisms through any of the various means known to those skilled in the art, a procedure that is called transformation (see also Keown et al. (1990 ) Meth Enzymol 185: 527-537). For example, DNA constructs can be introduced into cells, either in culture or in organs of a plant using a variety of conventional techniques. For example, DNA constructs can be introduced directly into plant cells using ballistic methods, such as bombardment of DNA particles, or DNA construction can be introduced using techniques such as electroporation and microinjection of cells. Particle-mediated transformation techniques (also known as "biolistics") are described, for example, in Klein et al. (1987) Nature 327: 70-73; Vasil V et al. (1993) BiolTechnol 11: 1553-1558; and Becker D et al. (1994) PlantJ 5: 299-307. These methods involve the penetration of cells by small particles with nucleic acid into the matrix of small microspheres or particles, or on the surface. The PDS-1000 Biolistic Gene Gun (Biorad, Hercules, CA) uses helium pressure to accelerate DNA micro-carriers coated with gold or tungsten DNA towards target cells. The process is applicable to a wide range of tissues and cells in organisms, including plants. Other transformation methods are also known to those skilled in the art.
[0179] Microinjection techniques are used in the technique and described in scientific literature and patent literature. In the same way, the cell can be chemically permeabilized, for example, using polyethylene glycol, in such a way that DNA can enter the cell by diffusion. DNA can also be introduced by fusing protoplasts with other units containing DNA such as minicells, cells, lysosomes or liposomes. The introduction of DNA constructs using polyethylene precipitation
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50/71 glycol (PEG) is described in Paszkowski et al. (1984) EMBO J 3: 2717. The distribution of liposome-based genes is, for example, described in WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; US 5,279,833; WO 91/06309; and Feigner et al. (1987) Proc Natl Acad Sci USA 84: 7413-7414).
[0180] Another suitable method of introducing DNA is electroporation, where cells are reversibly permeabilized by an electrical pulse. Electroporation techniques are described in Fromm et al. (1985) Proc Natl Acad Sci USA 82: 5824. PEG-mediated transformation and electroporation of plant protoplasts are also discussed in Lazzeri P (1995) Methods Mol Biol 49: 95-106. The preferred general methods that can be mentioned are: calcium phosphate-mediated transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction and infection. Such methods are known to those skilled in the art and described, for example, in Davis et al., Basic Methods In Molecular Biology (1986). For a review of gene transfer methods for plants and cell cultures, see, Fisk et al. (1993) Scientia Horticulturae 55: 5-36 and Potrykus (1990) Cl BA Found Symp 154: 198.
[0181] The methods are known for the introduction and expression of heterologous genes in monocot and dicot plants. See, for example, US 5,633,446, US 5,317,096, US 5,689,052, US 5,159,135, and US 5,679,558; Weising et al. (1988) Ann. Rev. Genet. 22: 421 -477. The transformation of monocots in particular can use several techniques, including electroporation (for example, Shimamoto et al. (1992) Nature 338: 274-276; biolistic (for example, EP-A1 270,356); and Agrobacterium (for example, Bytebier et al. (1987) Proc Natl Acad Sci USA 84: 5345-5349).
[0182] In plants, the methods for transforming and regenerating plants from plant tissues or plant cells with which
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51/71 individuals versed in the technique are familiar and are used for transient or stable transformation. Suitable methods are especially a protoplast transformation by means of DNA absorption induced by polyethylene glycol, biological methods such as the gene gun (“particle bombardment” method), electroporation, the incubation of dry embryos in a solution containing DNA, sonication and microinjection, and the transformation of intact cells or tissues by micro- or macroinjection into tissues or embryos, tissue electroporation, or vacuum infiltration of seeds. In the case of injection or electroporation of DNA into plant cells, the plasmid used does not have to satisfy any particular requirements. Simple plasmids like those in the pUC series can be used. If intact plants need to be regenerated from the transformed cells, the presence of an additional selectable marker gene in the plasmid will be useful.
[0183] In addition to these "direct" transformation techniques, transformation can also be carried out by bacterial infection through Agrobacteríum tumefaciens or Agrobacteríum rhizogenes. These strains contain a plasmid (Ti or Ri plasmid). Part of this plasmid, called T-DNA (transferred DNA), is transferred to the plant according to the Agrobacteríum infection and integrated into the plant cell genome.
[0184] For Agrobacterium-mediated transformation of plants, a DNA construct of the invention can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of host A. tumefaciens will direct the insertion of a transgene and adjacent marker gene (s) (if present) in the plant cell's DNA when the cell is infected by the bacterium. Transformation techniques mediated by Agrobacteríum tumefaciens are well described in the scientific literature. See, for example, Horsch et al. (1984) Science 233: 496-498, Fraley et al. (1983) Proc
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Natl Acad Sci USA 80: 4803-4807, Hooykaas (1989) Plant Mol Biol 13: 327-336, Horsch RB (1986) Proc Natl Acad Sci USA 83 (8): 2571-2575), Bevans et al. (1983) Nature 304: 184-187, Bechtold et al. (1993) Comptes Rendus De LAcademie Des Sciences Series ll-Sciences De La Vie-Life Sciences 316: 1 1941 199, Valkens et al. (1988) Proc Natl Acad Sci USA 85: 5536-5540.
[0185] Preferably, a DNA construct of the invention is integrated into specific plasmids, either in a conductive or intermediate vector, or in a binary vector. If, for example, a Ti or Ri plasmid needs to be used for transformation, at least the right boundary, but in most cases the right and left boundary of the Ti or Ri plasmid T-DNA is attached to the expression to be introduced as a flanking region. Binary vectors are preferably used. Binary vectors are capable of replication in both E. coli and Agrobacterium. They typically contain a selectable marker gene and a linker or polylinker flanked by the right and left T-DNA flanking sequence. They can be directly transformed into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187). The selection marker gene allows the selection of transformed agrobacteria and, for example, the nptll gene, which confers resistance to kanamycin. Agrobacterium, which acts as a host organism in this case, must contain a plasmid with the vir region. The latter is required to transfer T-DNA to the plant cell. An Agrobacterium so transformed can be used to transform plant cells.
[0186] Many strains of Agrobacterium tumefaciens are capable of transferring genetic material - for example, a DNA construct according to the invention such as, for example, the EHA101 (pEHA101) strains (Hood EE et al. (1996) J Bacteriol 168 (3): 1291 -1301), EHA105 (pEHA105) (Hood et al. 1 993, Transgenic Research 2, 208-218), LBA4404 (pAL4404) (Hoekema et al. (1983) Nature 303: 179-181), C58C1 (pMP90) (Koncz and Schell (1 986) Mol Gen
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Genet 204,383-396) and C58C1 (pGV2260) (Deblaere et al. (1985) NuclAcids Res. 13, 4777-4788).
[0187] The agrobacterial strain employed for the transformation comprises, in addition to its disarmed Ti plasmid, a binary plasmid with the T-DNA to be transferred, which normally comprises a gene for the selection of transformed cells and a gene to be transferred . Both genes must be equipped with transcriptional and translational initiation and termination signals. The binary plasmid can be transferred in the agrobacterial strain, for example, through electroporation or other transformation methods (Mozo & Hooykaas (1991) Plant Mol Biol 16: 917-918). Generally, plant explants are co-cultured with the agrobacterial strain for two to three days.
[0188] A variety of vectors could, or can, be used. In principle, these differentiate between those vectors that can be used for Agrobacterium-mediated transformation or agro-infection, that is, which comprises a DNA construction of the invention in a T-DNA, which, in fact, allows for a stable integration of the T -DNA in the plant genome. In addition, vectors free of borderline sequence can be used, which can be transformed into plant cells, for example, through bombardment of particles, where they can lead to both transient and stable expression.
[0189] The use of T-DNA for the transformation of plant cells has been studied and described extensively (EP-A1 120 516; Hoekema, ln: The Binary Plant Vector System, Offset-drukkerij Kanters V.. V., Alblasserdam, chapter V ; Fraley et al. (1985) Crit Rev Plant Sci 4: 1-45 and An et al. (1985) EMBO J 4: 277287). Several binary vectors are known, some of which are commercially available, for example, pBIN19 (Clontech Laboratories, Inc. USA).
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54/71 [0190] To transfer DNA to the plant cell, plant explants are co-cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Starting from infected plant material (for example, leaves, roots or stem sections, but also protoplasts or plant cell suspensions), intact plants can be regenerated using a suitable medium that may contain, for example, antibiotics or biocides to select transformed cells. The obtained plants can then be screened for the presence of the introduced DNA, in this case a DNA construct according to the invention. Once the DNA has integrated into the host genome, the genotype in question is usually stable and the insertion in question is also found in subsequent generations. Typically, the integrated expression cassette contains a selection marker that confers resistance to a biocide (for example, a herbicide) or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin and similar to the transformed vegetable. The selection marker allows the selection of transformed cells (McCormicket al., Plant CellReports5 (1986), 81-84). The obtained plants can be cultivated and hybridized in a conventional manner. Two or more generations must be cultivated in order to ensure that the genomic integration is stable and heritable.
[0191] The aforementioned methods are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu, Academic Press (1993 ), 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225). The construct to be expressed is preferably cloned into a vector that is suitable for the transformation of Agrobacterium tumefaciens, for example, pBin 19 (Bevan et al. (1984) NuclAcids Res 12: 871 1).
[0192] The DNA construct of the invention can be used to confer desired characteristics on essentially any plant. a
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55/71 A person skilled in the art will recognize that after the construction of DNA is stably incorporated into transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any number of standard breeding techniques can be used, depending on the species to be bred.
[0193] Alternatively, the optimized endonucleases can be expressed transiently. The chimeric endonuclease can be transiently expressed as a DNA or RNA distributed in the target cell and / or it can be distributed as a protein. Distribution as a protein can be achieved with the help of cell-penetrating peptides or by fusion with SEciV signal peptides fused to chimeric nucleases or endonucleases, which mediate secretion from a distribution organism into a cell of a target organism , for example, from Agrobacterium rhizogenes or Agrobacterium tumefaciens to a plant cell.
Regeneration of Transgenic Plants [0194] Transformed cells, that is, those comprising the DNA integrated into the host cell's DNA, can be selected from non-transformed cells if a selectable marker is part of the introduced DNA. A marker can, for example, be any gene that is capable of conferring resistance to antibiotics or herbicides (for examples see above). Transformed cells that express such a marker gene are able to survive in the presence of concentrations of a suitable antibiotic or herbicide that kills an untransformed wild type. Once the transformed plant cell has been generated, an intact plant can be obtained using methods known to skilled individuals. For example, callus cultures are used as starting material. The formation of branches and roots can be induced in this cell biomass not yet differentiated in the known way. The branches obtained can be planted and cultivated.
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56/71 [0195] Transformed plant cells, derived by any of the previous transformation techniques, can be cultured to regenerate a complete plant that has the transformed genotype and, therefore, the desired phenotype. These regeneration techniques depend on the manipulation of certain phytohormones in a tissue culture medium, typically depending on a biocidal and / or herbicidal marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124176, Macmillian Publishing Company, New York (1983); and in Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, (1985). Regeneration can also be achieved from plant callus, explants, somatic embryos (Dandekar et al. (1989) J Tissue Cult Meth 12: 145; McGranahan et al. (1990) Plant Cell Rep 8: 512), organs, or parts of it. These regeneration techniques are generically described in Klee et al. (1987) Ann Rev Plant Physiol 38: 467-486.
Combination with other recombination enhancement techniques [0196] In an additional preferred embodiment, the effectiveness of the recombination system is increased by combining with systems that promote homologous recombination. These systems are described and cover, for example, the expression of proteins such as RecA or treatment with PARP inhibitors. It has been shown that homologous intrachromosomal recombination in tobacco plants can be increased using PARP inhibitors (Puchta H et al. (1995) Plant J. 7: 203-210). The use of these inhibitors, the rate of homologous recombination in the recombination cassette after induction of the double-stranded DNA strand of specific sequence, and therefore the effectiveness of excluding the transgene sequences, can be further increased. Several PARP inhibitors can be used for this
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57/71 purpose. Preferably, inhibitors include 3-aminobenzamide, 8-hydroxy-2-methylquinazolin-4-one (NU1025), 1.11 b-dihydro- (2H) benzopyran (4,3,2de) isoquinolin-3-one (GPI 6150), 5-aminoisoquino-linone, 3,4-dihydro- 5- (4- (1piperidinyl) butoxy) -1 (2H) -isoquinolinone, or the compounds described in WO 00/26192, WO 00/29384, WO 00 / 32579, WO 00/64878, WO 00/68206, WO 00/67734, WO 01/23386 and WO 01/23390.
[0197] Furthermore, it was possible to increase the frequency of several homologous recombination reactions in plants expressing the E. coli RecA gene (Reiss B et al. (1996) Proc Natl Acad Sei USA 93 (7): 3094-3098) . Likewise, the presence of the protein shifts the ratio between homologous and illegitimate DSB repairs in favor of homologous repair (Reiss B et al. (2000) Proc Natl Acad Sei USA 97 (7): 3358-3363). Reference can also be made to the methods described in WO 97/08331 to increase homologous recombination in plants. An additional increase in the effectiveness of the recombination system can be achieved by the simultaneous expression of the RecA gene or other genes that increase the efficiency of homologous recombination (Shalev G et al. (1999) Proc Natl Acad Sei USA 96 (13): 7398-402 ). The systems described above for promoting homologous recombination can also be advantageously employed in cases where the recombination construct must be introduced in a site-directed manner into the genome of a eukaryotic organism by means of homologous recombination.
Methods for Homologous Recombination and Targeted Mutation Using Optimized Endonucleases.
[0198] The present invention provides a method for homologous recombination of polynucleotides which comprises:
The. provide a competent cell for homologous recombination,
B. provide a polynucleotide that comprises a
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58/71 recombinant polynucleotide flanked by an A sequence and a B sequence,
ç. providing a polynucleotide comprising sequences A 'and B', which are long enough and homologous to sequence A and sequence B, to permit homologous recombination in said cell, and
d. providing an optimized endonuclease or an expression cassette encoding an optimized endonuclease,
and. combine b), c) and d) in said cell, and
f. detect the recombined polynucleotides from b) and c), or select or grow cells comprising recombinant polynucleotides from
b) and c).
[0199] In an embodiment of the invention, step e) leads to the exclusion of a polynucleotide comprised in the polynucleotide provided in step c).
[0200] In one embodiment of the invention, the excluded polynucleotide comprised in the polynucleotide provided in step c) encodes a marker gene or parts of a marker gene.
[0201] In one embodiment of the invention, the polynucleotide provided in step b) comprises at least one expression cassette.
[0202] In one embodiment of the invention, the polynucleotide provided in step b) comprises at least one expression cassette, leading to the expression of a selectable marker gene or a reporter gene.
[0203] In one embodiment of the invention, the polynucleotide provided in step b) comprises at least one expression cassette, leading to the expression of a selectable marker gene or a reporter gene and comprises at least one DNA recognition site or at least a chimeric recognition site.
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59/71 [0204] An additional embodiment of the invention provides a method for targeted mutation of polynucleotides comprising:
The. providing a cell comprising a polynucleotide comprising a l-Scel recognition site,
B. provide an optimized endonuclease, being able to cleave the chimeric recognition site of step a),
ç. combine a) and b) in said cell and
d. detecting mutated polynucleotides, or selecting cultured cells comprising mutated polynucleotides.
[0205] The invention provides in another embodiment a method for homologous recombination as described above or a method for targeted mutation of polynucleotides as described above, which comprises:
combine the optimized endonuclease and the Scel recognition site through the crossing of organisms, through the transformation of cells or through a SeclV peptide fused to the optimized endonuclease and put the cell comprising the Scel recognition site in contact with an organism that expresses the endonuclease optimized and expresses a SeclV transport complex capable of recognizing the SeclV peptide fused to the chimeric endonuclease.
Examples
General Methods:
[0206] Chemical synthesis of oligonucleotides can be performed, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896- 897). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, the transfer of nucleic acids to nitrocellulose and nylon membranes, the bonding
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60/71 of DNA fragments, transformation of E. coli cells, bacterial cultures, phage propagation and recombinant DNA sequence analysis are performed as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. The recombinant DNA molecules were sequenced using an ALF Express fluorescent laser DNA sequencer (Pharmacia, Upsala [sic], Sweden) following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
Example 1: Constructions That Include Sequence-Specific DNA Endonuclease Expression Cassettes for Expression in E.cou
Example 1 a: Basic Construction [0207] In this example, a general outline of a vector is presented, called "Construction I" suitable for transformation into E. coli. This general outline of the vector comprises an ampicillin-resistant gene for selection, an origin of replication for E. coli and the araC gene, which encodes an Arabinose-inducible transcriptional regulator. SEQ ID NO: 7 shows a sequence expansion of “NNNNNNNNNN”. This means a placeholder for genes that encode different versions of the sequence-specific DNA endonuclease. The different genes can be expressed from the Arabinose-inducible pBAD promoter (Guzman et al., J Bacteriol TT. 4121-4130 (1995)), the sequences of the genes encoding the different versions of nuclease are given in the examples to follow.
[0208] The control construct, in which the placeholder is replaced by the l-Scel sequence (SEQ ID NO: 8), was referred to as VCSAH40-4.
Example 2: E. coli Plasmids Encoding Stabilized Versions of Nuclease [0209] The different destabilizing sequences can be identified in the amino acid sequence of l-Scel. Among these, one
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61/71 weak PEST sequence at the C-terminal, which comprises amino acid residues 228 to 236 and an N-terminal sequence that shows similarity to a KEN motif (Pflegere Kirschner, Genes andDev. 14: 655-665 (2000)). According to the N-terminal rule, the second amino acid residue of l-Scel gives instability to the protein.
[0210] To test the effect of these sequences on nuclease stability, different versions of l-Scel were generated by PCR, which lacks N-terminal amino acids, 9 C-terminal amino acids or both. These constructions were expressed from “Construction I”, described in Example 1
The). Therefore, the placeholder was replaced by several sequences, encoding the nuclease versions (shown in SEQ ID NO: 2, 3, 5). The plasmids were named VC-SAH43-8 (Cterminal shortened l-Scel) and VC-SAH42-13 (NLS-C shortened l-Scel), VC-SAH44- 32 (N-terminal shortened IScel, SEQ ID NO: 21) and VC-SAH45-3 (Ne C-terminal shortened l-Sce, SEQ ID NO: 22).
[0211] According to the N-terminal rule, all of these constructs lead to the stabilization of the second amino acid G residue. To test the effect of the second amino acid on protein stability, versions are also generated with the native destabilizing residue of l- Scel. The resulting plasmids were named VC-SAH105 and VC-SAH106.
[0212] Additional exclusions from the C-terminal were generated:
the unique amino acid residues were successively removed from the C-terminal. These variants are summarized in Table 3) and were tested to determine their activity in E.coli.
[0213] In addition, potential PEST sequences were discovered in l-Scel and analyzed by introducing unique amino acid exchanges. These variants are summarized in Table 3) and were tested to determine their activity in E.coli.
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Table 3
Vector name Nuclease variant VC-SAH 151-2 NLS l-Scel -1 VC-SAH 152-6 NLS l-Scel -2 VC-SAH 153-6 NLS l-Scel -3 VC-SAH 154-1 NLS l-Scel -4 VC-SAH 155-1 NLS l-Scel -5 VC-SAH 156-3 NLS l-Scel -6 VC-SAH 157-1 NLS l-Scel -7 VC-SAH 158-2 NLS l-Scel -8 VC-SAH 159-3 NLS l-Scel -10 VC-SAH 160-1 NLS l-Scel -11 VC-SAH 161-1 NLS l-Scel -12 VC-SAH 162-2 NLS l-Scel -13 VC-SAH 163-1 NLS l-Scel 1-218 VC-SAH 164-2 NLS l-Scel 1 -202 VC-SAH 165-3 NLS l-Scel 1 -187 VC-SAH 166-1 NLS l-Scel 1 -169 VC-SAH 167-1 NLS l-Scel 1 -155 VC-SAH 190-4 l-Scel L74K VC-SAH 191 -3 l-Scel Y75H VC-SAH 192-3 l-Scel Q77K VC-SAH 193-3 -Scel E130K VC-SAH 194-1 l-Scel T134H VC-SAH 195-2 l-Scel Y199H VC-SAH 196-2 l-Scel M203K VC-SAH 197-2 l-Scel Y205H VC-SAH 198-1 l-Scel S230K
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Example 3: Co-Transformation of Constructs Encoding DNA Endonuclease and Constructions Covering Nuclease Recognition Sequences in E. coli [0214] Plasmids VC-SAH44-32, VC-SAH43-8, VCSAH42-13, VC-SAH45- 3 and VC-SAH40-4 (described in Example 2) were individually co-transformed with the target vector VC-SAH6-1 or with the control vector VC-SAH7-1 in E. coli. The same is done with VC-SAH 105 and VC-SAH 106 and with the vectors summarized in Table 3.
Example 4: Demonstration of Endonuclease Activity in E. coli [0215] The versions of l-Scel described in Example 2 were tested to determine their activity.
[0216] Co-transformants conducting the combination of two plasmids, one encoding a nuclease and the other including the nuclease target site were grown overnight in LB with Ampicillin, Kanamycin and Glucose to suppress the pBAD promoter. Cultures were diluted 1: 100 and grown until OD6oo = 0.5 was reached. Nuclease expression was induced by the addition of Arabinose for 3 to 4 hours. The pBAD promoter is described as being dose dependent (Guzman 1995), therefore, the culture was divided into different aliquots and protein expression was induced with Arabinose concentrations ranging from 0.2% to 0.0002%. 5 pm of each aliquot were plated in a solid LB medium, supplemented with Ampicillin and Kanamycin. The plates were incubated overnight at 37 C and ^S cell growth was analyzed semi-quantitatively. Active nuclease fusions did not cut the constructions, which span the target site. This leads to loss of resistance to kanamycin. Therefore, the activity of the fusion protein was observed due to the loss capacity of the co-transformants for cultivation in a medium containing kanamycin.
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Results [0217] VC-SAH43-8 (C-terminal shortened l-Scel) and VC-SAH42-13 (NLS-C shortened l-Scel) were very active, cutting the target site even in the absence of the Arabinose inductor. Cell culture of these cotransformants was observed only in the presence of Glucose, which further suppresses the pBAD promoter. Therefore, in the cases of VC-SAH43-8 and VCSAH42-13, the low amount of l-Scel protein produced due to basal expression from the pBAD promoter was sufficient to cut the target plasmid.
[0218] The results are simplified and summarized in Table 4 ++ and + represent a very strong and strong growth, which indicates little or no nuclease activity expressed towards the respective target site. - and - - represent little or no growth, which indicates a high or very high activity of the nuclease towards the respective target site.
Table 4: Variants l-Scel: The E. col Growth Test / Indicates Endonuclease Activity in Relation to the respective Target Sites
Nuclease variant VC-SAH6-1 (site l-Scel) VC-SAH7-1 (control) VC-SAH40-4 l-Scel + ++ VC-SAH43-8 l-Scel short C-term (-9) l-Scel short NLS -C term - + VC-SAH42-13 (-9) - + VC-SAH151-2 NLS l-Scel -1++ VC-SAH 152-6 NLS l-Scel -2++ VC-SAH 153-6 NLS l-Scel -3++ VC-SAH 154-1 NLS l-Scel -4++ VC-SAH 155-1 NLS l-Scel -5++ C-SAH 156-3 V NLS l-Scel -6++ VC-SAH 157-1 NLS l-Scel -7+ VC-SAH 158-2 NLS l-Scel -8+ VC-SAH 159-3 NLS l-Scel-10+ VC-SAH 160-1 NLS l-Scel -11 ++ ++ VC-SAH 161-1 NLS l-Scel-12 ++ ++ VC-SAH 162-2 NLS l-Scel-13 ++ ++ VC-SAH 163-1 NLS l-Scel 1-218 ++ ++ VC-SAH 164-2 NLS l-Scel 1-202 ++ ++ VC-SAH 165-3 NLS l-Scel 1-187 ++ ++
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Nuclease variant VC-SAH6-1 (site l-Scel) VC-SAH7-1 (control) VC-SAH 166-1 NLS l-Scel 1-169 ++ ++ VC-SAH 167-1 NLS l-Scel 1-155 ++ ++
Example 5: Transformation of S. cerevisiae [0219] S. cerevisiae cells are grown in 10 ml of YEPS overnight and then diluted 1:10. This culture is then grown until OD6oo = 0.5 is reached. The cells are pelleted and resuspended in 15 ml of sterile water twice, again pelleted and resuspended in 1 ml of sterile water. This cell suspension is aliquoted at 100 μΙ and pelleted again. On ice, 240 μΙ of 50% PEG4000, 36 μΙ of 1 M LiAc, 20 μΙ of salmon sperm DNA (5 mg / ml) were added (5 minutes at 100 ^S C, then 10 minutes on ice ) and 6 pg of plasmid in 64 μΙ of water. The suspension is incubated at 42 ^O C for 45 minutes and placed on ice for 30 seconds. The cells are pelleted and resuspended in 500 μΙ of water, of which 200 μΙ are plated in a selective medium without methionine. The plates are incubated at 30 ^O C for 3 to 4 days. Single colonies can be chosen for future analysis.
Example 6: Constructions Spanning Stabilized Versions of Nuclease for Expression in S. cerevisiae [0220] The sequences described in Example 2 are cloned into the vector pGBT9-3H / B (Tirode et al 1997, J Biol Chem 272: 22995-22999) under the control of the MET25 promoter, which is represented in the presence and active in the absence of methionine.
Example 7: Demonstration of Endonuclease Stability in S. CEREVISIAE [0221] Protein expression is induced by the growth of transformants in a medium lacking methionine. The complete protein extract from the different transformants is generated and tested for determination
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66/70 of the abundance and amount of l-Scel by Western blot analysis. Pulse-seeking experiments (from English "pulse-chase") were carried out using Cicloeximide and MG132, to determine the in vivo half-life of the different versions.
Example 8: Constructions that Encode Stabilized Versions of Nuclease for Expression in A. thaliana
Example 8 a: Constructions for Demonstrating Endonuclease Activity Crossing Plants Expressing Nuclease with Plants Conducting a T-DNA with Their Target Site [0222] All constructs showing activity in Table 4 are evaluated by testing , the following Examples will focus on the shortened C-terminal version of l-Scel. Different plasmids were generated, where the "Construction IV" position marker (SEQ ID No: 13) is replaced by different sequences, encoding the shortened C-terminal version of l-Scel, in combinations with or without stabilizing G such as second amino acid residue, and with or without NLS. The nuclease variants are more favorable among the constructs VC-SAH151 -2, VC-SAH152-6, VC-SAH153-6, VC-SAH 154-1, VC-SAH 155-1, VC-SAH 156-3.
Example 8 b: Constructions for Demonstration of Endonuclease Activity Transforming These Constructions into Plants that already Conduct a T-DNA with the respective Target Site [0223] In this example, we present the general outline of a binary vector, denominated as “ Construction VI ”(VC-SCB697) suitable for vegetable processing. This general outline of the binary vector comprises a TDNA with a cassette nos-promoter :: nptll :: nos-terminator, which allows selection in kanamycin when integrated into the plant genome. SEQ ID NO: 23 (VCSCB697) shows a sequence extension of "NNNNNNNNNN". This means a placeholder for genes that encode versions of l-Scel.
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67/70 [0224] Different plasmids were generated, where the position marker is replaced by different constructions, which consist of a shortened C-terminal version of l-Scel: VC-SAH124-3 (N shortened LS-l-Scel C term, G) (SEQ ID NO: 5), VC-SAH125-2 (shortened l-Scel C term, G), (SEQ ID NO: 3), VC-SAH122-7 (l-Scel, G) ( SEQ ID NO: 2) and VC-SAH123-3 (NLS-l-Scel, G), see Example 2 (Used as a l-Scel control without the stabilizing G as the second amino acid residue: VC-SCB697-3 ). All constructs showing activity in Table 4 are evaluated by test, the nuclease variants encoded by constructions VC-SAH151 -2, VC-SAH 152-6, VC-SAH 153-6, VC-SAH 154-1, VC- SAH 155-1, VC-SAH 156-3 are the most favorable.
[0225] Identical plasmids are generated without the stabilizing G as the second amino acid residue.
Example 9: Transformation of Constructions Encoding Stabilized Versions of Nuclease into A. thaliana [0226] The plasmids described in Example 8b were transformed into A. thaliana strains that carry the VCSCB583-40 T-DNA (SEQ ID NO: 24) .
[0227] The constructions described in Example 8a) are transformed into wild type plants.
Example 10: Monitoring the Activity of Stabilized Nucleases Example 10a: by Crossing [0228] The activity of the different versions of l-Scel is monitored by crossing the specific sequence DNA endonuclease expression lines and the lines that comprise constructions with sequences recognition. The recognition sequences are surrounded by a partial uidA (GUS) gene (called “GU”) and another partial uidA gene (called “US”). The partially overlapping halves of the GUS gene (GU and US) are non-functional, however, as a result of the activity of l-Scel in the
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68/70 target site, a functional GUS gene will be restored by homologous intrachromosomal recombination (ICHR). This can be monitored by a histochemical assay for GUS placement by Jefferson et al. (1987) EMBO J 6: 3901 3907).
[0229] To view l-Scel activity, the Arabidopsis transgenic strains that span the T-DNA of the plasmids described in Example 9a) are crossed with the Arabidopsis strains that span the VC-SCB734-4 construction T-DNA. . F1 seeds from crosses are harvested. The seeds have their surfaces sterilized and grown in medium A supplemented with the respective antibiotics and / or herbicides. The 3-4 days old seedlings are harvested and used for the GUS staining histochemical assay. The amount of blue areas is an indicator of tissues / tissue parts where ICHR occurred at crossings and, therefore, for l-Scel activity.
Example 10b: by Supertransformation [0230] The activity of the different versions of l-Scel was monitored by transforming the lines that comprise the constructions with plasmid recognition sequences that cover an expression cassette with different versions of the stabilized l-Scel. The recognition sequences are surrounded by a partial uidA (GUS) gene (called “GU”) and another partial uidA gene (called “US”). The partially overlapping halves of the GUS gene (GU and US) are non-functional, however, as a result of l-Scel activity at the target site, a functional GUS gene will be restored by homologous intrachromosomal recombination (ICHR). This can be monitored by a histochemical assay for GUS placement by Jefferson et al. (1987) EMBO J 6: 3901-3907).
[0231] To view the activity of l-Scel, the Arabidopsis transgenic strains that comprise the construction T-DNA pCB583-40
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69/70 were transformed with plasmids described in Example 8b). F1 seeds were harvested, their surfaces sterilized and grown in medium A supplemented with the respective antibiotics and / or herbicides. F1 plants were analyzed for single copy integration of the nuclease construct and self-reproduced. F2 plants were grown in medium A without selection pressure. The T-DNA that encodes the nuclease also encodes dsRed. Due to dsRed-free segregation and, therefore, nuclease-free plants were selected under UV light. The 4-leaf seedlings were harvested and used for the GUS staining histochemical assay. The blue seedlings represent a homologous recombination event, which occurred in the previous generation. For each construction, 3 to 5 independent strains were analyzed, up to 96 seedlings were stained. The number of blue seedlings is an indicator for l-Scel activity.
Results [0232] In summary, l-Scel, l-Scel + G and N LS-l-Scel + G resulted in between 30% -41% of blue plants. While the expression of the shortened C-terminal versions encoded by VC-SAH124-3 and VC-SAH 125-2 resulted in approximately 60% of blue seedlings.
[0233] A positive GUS signal represents an ICHR event, due to the activity of l-Scel. The nuclease can also produce a cut, which may not be repaired by ICHR, but by illegitimate recombination. This event will lead to the destruction of the l-Scel recognition sequence and a non-functional GUS gene. In this case, the activity of l-Scel may not be monitored by the blue tint. To further analyze the white seedlings obtained in this assay, a PCR reaction was performed that amplifies the halves of the GUS gene (GU and US). The amplicons were subjected to l-Scel digestion to detect the presence or absence of the target sequence. The absence of the target site represents the activity of l-Scel in the previous generation. In summary, the shortened C-terminal l-Scel variants resulted in 1 out of 88 T2 plants tested with one site
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70/70 l-Scel intact. In contrast, the l-Scel encoded by the VC-SCB697-3 construct resulted in 14 out of 48 plants tested that still covered an uncut l-Scel site.
[0234] The shortened C-terminal versions encoded by VC-SAH 124-3 and VC-SAH 125-2 resulted in a T2 generation in which almost all individuals showed the result of l-Scel activity.

权利要求:
Claims (5)
[1]
Claims
1. ENDONUCLEASE, characterized by consisting of an amino acid sequence described by SEQ ID NO: 3 or 5.
[2]
2. ENDONUCLEASE, according to claim 1, characterized by being an endonuclease produced by genetic engineering.
[3]
3. METHOD FOR HOMOLOGICAL RECOMBINATION OF POLYNUCLEOTIDS, characterized by comprising:
a) provide a competent cell for homologous recombination,
b) providing a polynucleotide comprising an endonuclease DNA recognition site flanked by an A sequence and a B sequence,
c) providing a polynucleotide comprising sequences A 'and B', having a length of 15 to 30 nucleotides and which are homologous to sequence A and sequence B, and
d) providing an endonuclease, as defined in any one of claims 1 to 2,
e) combine b), c) and d) in said cell, and
f) detecting recombined polynucleotides from b) and c), or selecting or culturing cells comprising recombinant polynucleotides from b) and c).
[4]
METHOD according to claim 3, characterized in that through homologous recombination a polynucleotide sequence comprised in the competent cell of step a) is excluded from the genome of the culturing cells of step f).
[5]
5. METHOD FOR DIRECTED MUTATION OF POLYNUCLEOTIDS, characterized by comprising:
a) providing a cell that comprises a polynucleotide
Petition 870180143725, of 10/23/2018, p. 81/90
2/2 comprising an endonuclease DNA recognition site, as defined in any one of claims 1 to 2,
b) providing an endonuclease, as defined in any one of claims 1 to 2, and which is capable of cleaving the DNA recognition site of step a),
c) combining a) and b) in said cell, and
d) detecting mutated polynucleotides, or selecting or culturing cells comprising mutated polynucleotides.

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同族专利:

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JP2013511977A|2013-04-11|

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ZA201204700B|2013-09-25|

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EP2504439A4|2013-06-12|

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法律状态:
2018-02-06| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2018-08-21| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2019-02-19| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-03-26| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/11/2010, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/11/2010, OBSERVADAS AS CONDICOES LEGAIS |

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