(en)An I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG (SEQ ID NO: 229) core domain situated from positions 26 to 40 and 44 to 77 of I-CreI, said variant being able to cleave a DNA target sequence from a xeroderma pigmentosum gene. Use of said variant and derived products for the prevention and the treatment of Xeroderma pigmentosum.

1.ApplicationNumber: US-27924507-A
1.PublishNumber: US-2009222937-A1
2.Date Publish: 20090903
3.Inventor: ARNOULD SYLVAIN
PEREZ-MICHAUT CHRISTOPHE
SMITH JULIANNE

4.Inventor Harmonized: ARNOULD SYLVAIN(FR)
PEREZ-MICHAUT CHRISTOPHE(FR)
SMITH JULIANNE(FR)

5.Country: US
6.Claims:
(en)An I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG (SEQ ID NO: 229) core domain situated from positions 26 to 40 and 44 to 77 of I-CreI, said variant being able to cleave a DNA target sequence from a xeroderma pigmentosum gene. Use of said variant and derived products for the prevention and the treatment of Xeroderma pigmentosum.

7.Description:
(en)The invention relates to a meganuclease variant cleaving a DNA target sequence from a xeroderma pigmentosum gene (XP gene), to a vector encoding said variant, to a cell, an animal or a plant modified by said vector and to the use of said meganuclease variant and derived products for genome therapy, in vivo and ex vivo (gene cell therapy), and genome engineering.
Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disease characterized by a hypersensitivity to exposure to ultraviolet A (UV) rays, a high predisposition for developing skin cancers on sunlight exposed areas, and in some cases neurological disorders (Hengge, U. R. and W. Bardenheuer, Am. J. Med. Genet. C. Semin. Med. Genet., 2004, 131: 93-100; Magnaldo, T. and A. Sarasin, Cells Tissues Organs, 2004, 177: 189-198; Cleaver, J. E., Nat. Rev. Cancer, 2005, 5: 564-573; Hengge U. R., Clin. Dermatol., 2005, 23: 107-114). Cells of XP patients present a reduced capacity to eliminate UV induced DNA lesions (Cordonnier, A. M. and R. P. Fuchs, Mutat. Res., 1999, 435, 111-119). Such abnormality results from a defect in the Nucleotide Excision Repair (NER) process, a versatile mechanism conserved among eukaryotes and implicated in the correction of the damaged DNA by excision of the damaged nucleotides and re-synthesis. Defect in this process leads to a persistence of UV damage in the DNA, resulting in mutagenesis and tumour development in the UV exposed skin area. The three major types of skin cancers, squamous cell carcinomas, basal cell carcinomas and malignant melanomas already appear in childhood. XP Patients were assigned to 7 complementation groups (XP-A to XP-G) by cell fusion experiments, and each complementation group turned out to result from mutations in a distinct NER gene. The human genes, and the encoded proteins were often named after the complementation group. For example, the XPC gene ( FIG. 1A ), mutated in the XP-C complementation group, codes for a DNA damage binding protein.
Until now the only treatment available to XP patients is either full protection against sun exposure (as well as against certain common lamps producing long-wavelength UV) or repeated surgery to remove appearing skin cancers. Several attempts of autologous graft have been made to replace such cancerous area with skin from unexposed parts of the patient's body. However, since the grafted cells are also sun-sensitive the benefits for the patients are at best, limited to a few years, and the majority of patients die before reaching adulthood because of metastases. Skin engraftment can be made locally, but with the general limitations of grafts, in term of immunological tolerance.
Thus gene and cell therapy represent a huge hope for this kind of disease. Since cells from the skin lineage can be easily manipulated in vitro, a possibility would be to manipulate patient cells and correct their genetic defect, before grafting them back at the site of the tumour. Compared to other XP complementation groups, XP-C seems to be the best candidate for corrective gene transfer. In Europe and North Africa, XP-C is involved in more than half of the XP patients and although XPC expression is ubiquitous, XP-C patients remain free of neurological problems observed in other XP groups. Preliminary studies aimed at tissue therapy of XP patients have shown that in vitro retroviral transduction of XP fibroblasts from various complementation groups (XP-A, XP-B, XP-C, XP-D) and of XP-C primary keratinocytes with the XP cloned genes result in the recovery of full DNA repair capacity (Arnaudeau-Begard et al., Hum. Gene Ther., 2003, 14, 983-996; Armelini et al., Cancer Gene Ther., 2005, 12, 389-396). Furthermore, cells from the skin lineage can be easily manipulated, and then used to reconstruct functional skin (Amaudeau-Begard et al., Hum. Gene Ther., 2003, 14, 983-996; Armelini et al., Cancer Gene Ther., 2005, 12, 389-396). Thus, a rationale and promising alternative for long term tissular therapy would then consist in an ex vivo gene correction of the XP-C locus in keratinocytes before grafting back a reconstructed skin to the patient.
Homologous recombination is the best way to precisely engineer a given locus. Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M. R., Science, 1989, 244: 1288-1292; Smithies O., Nat. Med., 2001, 7: 1083-1086) or knock-in exogenous sequences in the chromosome.
It can as well be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases, such as XP. However, this application is in fact difficult, due to the low efficiency of the process (10 −6 to 10 −9 of transfected cells). In the last decade, several methods have been developed to enhance this yield. For example, chimeraplasty (De Semir et al. J. Gene Med., 2003, 5: 625-639) and Small Fragment Homologous Replacement (Goncz et al., Gene Ther, 2001, 8: 961-965; Bruscia et al., Gene Ther., 2002, 9: 683-685; Sangiuolo et al., BMC Med. Genet., 2002, 3: 8-; De Semir and Aran, Oligonucleotides, 2003, 13: 261-269; U.S. Pat. No. 6,010,908) have both been used to try to correct CFTR mutations with various levels of success.
Another strategy to enhance the efficiency of recombination is to deliver a DNA double-strand break in the targeted locus, using meganucleases. Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (12 to 45 bp). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21: 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-5060; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-277; Donoho et al., Mol. Cell. Biol, 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448). Recently, I-SceI was used to stimulate targeted recombination in mouse hepatocytes in vivo. Recombination could be observed in up to 1% of hepatocytes (Gouble et al., J. Gene Med., 2006, 8, 616-622).
However, the use of this technology is limited by the repertoire of natural meganucleases. For example, there is no cleavage site for a known natural meganuclease in human XP genes. Therefore, the making of meganucleases with tailored specificities is under intense investigation, and several laboratories have tried to alter the specificity of natural meganucleases or to make artificial endonuclease.
Recently, fusion of Cys2-His2 type Zinc-Finger Proteins (ZFP) with the catalytic domain of the Type IIS FokI endonuclease were used to make functional sequence-specific artificial endonucleases (Smith et al., Nucleic Acids Res., 1999, 27: 674-681; Bibikova et al., Science, 2003, 300: 764; Porteus M. H. and D. Baltimore, Science, 2003, 300: 763). The binding specificity of ZFPs is relatively easy to manipulate, and a repertoire of novel artificial ZFPs, able to bind many (g/a)nn(g/a)nn(g/a)nn sequences is now available (Pabo et al., Annu. Rev. Biochem., 2001, 70, 313-340; Segal, D. J. and C. F. Barbas, Curr. Opin. Biotechnol., 2001, 12, 632-637; Isalan et al., Nat. Biotechnol., 2001, 19, 656-660). This last strategy allowed recently for the engineering of the IL2RG gene in vitro (Urnov et al., Nature, 2005, 435, 646-651). Nevertheless, preserving a very narrow specificity is one of the major issues for genome engineering applications, and presently it is unclear whether ZFPs would fulfill the very strict requirements for therapeutic applications.
Homing Endonucleases (HEs) are a widespread family of natural meganucleases including hundreds of proteins (Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). These proteins are encoded by mobile genetic elements which propagate by a process called “homing”: the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus (Kostriken et al., Cell; 1983, 35, 167-174; Jacquier, A. and B. Dujon, Cell, 1985, 41, 383-394). Given their natural function and their exceptional cleavage properties in terms of efficacy and specificity, HEs provide ideal scaffolds to derive novel endonucleases for genome engineering. Data have been accumulated over the last decade, characterizating the LAGLIDADG family, the largest of the four HE families (Chevalier and Stoddard, precited). LAGLIDADG refers to the only sequence actually conserved throughout the family and is found in one or (more often) two copies in the protein. Proteins with a single motif, such as I-CreI, form homodimers and cleave palindromic or pseudo-palindromic DNA sequences, whereas the larger, double motif proteins, such as I-SceI are monomers and cleave non-palindromic targets. Seven different LAGLIDADG proteins have been crystallized, and they exhibit a very striking conservation of the core structure, that contrasts with the lack of similarity at the primary sequence level (Jurica et al., Mol. Cell., 1998, 2, 469-476; Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier et al., J. Mol. Biol., 2003, 329, 253-269; Moure et al., J. Mol. Biol, 2003, 334, 685-695; Moure et al., Nat. Struct. Biol., 2002, 9, 764-770; Ichiyanagi et al., J. Mol. Biol., 2000, 300, 889-901; Duan et al., Cell, 1997, 89, 555-564; Bolduc et al., Genes Dev., 2003, 17, 2875-2888; Silva et al., J. Mol. Biol., 1999, 286, 1123-1136). In this core structure, two characteristic αββαββα folds, also called LAGLIDADG Homing Endonuclease Core Domains, contributed by two monomers, or by two domains in double LAGLIDAG proteins, are facing each other with a two-fold symmetry. DNA binding depends on the four β strands from each domain, folded into an antiparallel β-sheet, and forming a saddle on the DNA helix major groove ( FIG. 2 ). Analysis of I-CreI structure bound to its natural target shows that in each monomer, eight residues (Y33, Q38, N30, K28, Q26, Q44, R68 and R70) establish direct interactions with seven bases at positions ±3, 4, 5, 6, 7, 9 and 10 (Jurica et al., 1998, precited; FIG. 3 ). In addition, some residues establish water-mediated contact with several bases; for example S40 and N30 with the base pair at position +8 and −8 (Chevalier et al., 2003, precited). The catalytic core is central, with a contribution of both symmetric monomers/domains. In addition to this core structure, other domains can be found: for example, PI-SceI, an intein, has a protein splicing domain, and an additional DNA-binding domain (Moure et al., 2002, precited; Grindl et al., Nucleic Acids Res., 1998, 26, 1857-1862).
Two approaches have been used to derive novel endonucleases with new specificities, from Homing Endonucleases:
Protein Variants
Seligman and co-workers used a rational approach to substitute specific individual residues of the I-CreI αββαββα fold (Sussman et al., J. Mol. Biol., 2004, 342, 31-41; Seligman et al., Genetics, 1997, 147, 1653-64); substantial cleavage was observed for few I-CreI variants (Y33C, Y33H, Y33R, Y33L, Y33S, Y33T, S32K, S32R) and only for a target modified in position ±10.
In a similar way, Gimble et al. modified the additional DNA binding domain of PI-SceI (J. Mol. Biol., 2003, 334, 993-1008); they obtained protein variants with altered binding specificity but no altered specificity and most of the variants maintained a lot of affinity for the wild-type target sequence.
The semi-rational approach used in theses studies permits the identification of endonucleases with altered specificity; however, it does not allow the direct production of endonucleases with predicted specificity.
Hybrid or Chimeric Single-Chain Proteins
New meganucleases could be obtained by swapping LAGLIDADG Homing Endonuclease Core Domains of different monomers (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). These single-chain chimeric meganucleases wherein the two LAGLIDADG Homing Endonuclease Core Domains from different meganucleases are linked by a spacer, are able to cleave the hybrid target corresponding to the fusion of the two half parent DNA target sequences.
The construction of chimeric and single chain artificial HEs has suggested that a combinatorial approach could be used to obtain novel meganucleases cleaving novel (non-palindromic) target sequences: different monomers or core domains could be fused in a single protein, to achieve novel specificities. These results mean that the two DNA binding domains of an I-CreI dimer behave independently; each DNA binding domain binds a different half of the DNA target site ( FIG. 2A ). Recently, a two steps strategy was used to tailor the specificity of a natural HEs such as I-CreI (Arnould et al., J. Mol. Biol., 2006, 355: 443-458). In a first step, residues Q44, R68 and R70 were mutagenized, and a collection of variants with altered specificity in positions ±3 to 5 (5NNN DNA target) were identified by screening. In a second step, two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of a different half of each variant DNA target sequence.
The generation of collections of novel meganucleases, and the ability to combine them by assembling two different monomers/core domains considerably enriches the number of DNA sequences that can be targeted ( FIG. 4A ), but does not yet saturate all potential sequences.
To reach a larger number of sequences, it would be extremely valuable to be able to identify smaller independent subdomains that could be combined ( FIG. 2B ).
However, a combinatorial approach is much more difficult to apply within a single monomer or domain than between monomers since the structure of the binding interface is very compact and the two different ββ hairpins which are responsible for virtually all base-specific interactions do not constitute separate subdomains, but are part of a single fold. For example, in the internal part of the DNA binding regions of I-CreI, the gtc triplet is bound by one residue from the first hairpin (Q44), and two residues from the second hairpin (R68 and R70; see FIG. 1B of Chevalier et al., 2003, precited).
A semi rational design assisted by yeast high throughput screening method allowed the Inventors, to identify and isolate thousands of I-CreI variants in positions 28, 30, 33, 38 and 40 with altered specificities in positions ±8 to 10 (10NNN DNA target). These new proteins were designed to cleave one of the 64 targets degenerate at nucleotides ±10, ±9, ±8 (10NNN DNA target) of the I-CreI original target site ( FIG. 3 ). Furthermore, in spite of the lack of apparent modularity at the structural level, residues 28 to 40 binding to positions ±8 to 10 and residues 44 to 77 binding to positions ±3 to 5 of the I-CreI site, were revealed to form two separable functional subdomains, able to bind distinct parts of an I-CreI homing endonuclease half-site ( FIG. 3 and FIG. 4B ). By assembling two subdomains from different monomers or core domains within the same monomer, the Inventors have engineered functional homing endonuclease (homodimeric) variants, which are able to cleave palindromic chimeric targets ( FIG. 4B ) having the nucleotides in positions ±3 to 5 and ±8 to 10 of each parent monomer/core domain. Furthermore, a larger combinatorial approach is allowed by assembling four different subdomains ( FIG. 4C : top right, middle left and right, bottom left) to form new heterodimeric molecules which are able to cleave non-palindromic chimeric targets (bottom right). The different subdomains can be modified separately and combine in one meganuclease variant (heterodimer or single-chain molecule) which is able to cleave a target from a gene of interest. The engineered variant can be used for gene correction via double-strand break induced recombination ( FIGS. 1B and 1C ).
The capacity to combine four sub-domains considerably increases the number of DNA sequences that can be targeted ( FIG. 4C ). However, it is still difficult to fully appreciate the range of sequences that can be reached with this combinatorial approach. One of the most elusive factors is the impact of the four central nucleotides of the I-CreI target site (gtac in the palindromic I-CreI site C1221, FIG. 3 ). Even though the base-pairs ±1 and ±2 do not display any contact with the protein, it has been shown that these positions are not devoid of content information (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269), especially for the base-pair ±1 and could be a source of additional substrate specificity (Argast et al., J. Mol. Biol., 1998, 280, 345-353; Jurica et al., Mol. Cell., 1998, 2, 469-476; Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). In vitro selection of cleavable I-CreI target (Argast et al., precited) randomly mutagenized, revealed the importance of these four base-pairs on protein binding and cleavage activity. It has been suggested that the network of ordered water molecules found in the active site was important for positioning the DNA target (Chevalier et al., Biochemistry, 2004, 43, 14015-14026). In addition, the extensive conformational changes that appear in this region upon I-CreI binding suggest that the four central nucleotides could contribute to the substrate specificity, possibly by sequence dependent conformational preferences (Chevalier et al., 2003, precited).
Thus, it was not clear if mutants identified on 10NNN and 5NNN DNA targets as homodimers cleaving a palindromic sequence with the four central nucleotides being gtac, would allow the design of new endonucleases that would cleave targets containing changes in the four central nucleotides.
The Inventors have identified hundreds of DNA targets in the XP genes that could be cleaved by I-CreI variants. The combinatorial strategy described in FIG. 4 was used to extensively redesign the DNA binding domain of the I-CreI protein and thereby engineer novel meganucleases with fully engineered specificity, to cleave two DNA targets from the XPC gene (Xa.1 and Xc.1) which differ from the I-CreI C1221 22 bp palindromic site by 17 nucleotides including the four central nucleotides in positions ±1 to 2 (Xa.1, FIGS. 3 , 9 and 23 ) or 11 nucleotides including two (positions −1 and −2) of the four central nucleotides (Xc.1, FIG. 23 ).
Even though the combined variants were initially identified towards nucleotides 10NNN and 5NNN respectively, and a strong impact of the four central nucleotides of the target on the activity of the engineered meganuclease was observed, functional meganucleases with a profound change in specificity regarding the other base-pairs of the target were selected. Furthermore, the activity of the engineered protein could be significantly improved by two successive rounds of random mutagenesis and screening, to compare with the activity of the I-CreI protein. Finally, the extensive redesign of the DNA binding domain is not made at the expense of the level of specificity, the novel endonucleases keeping a very narrow numbers of cleavable cognate targets.
These I-CreI variants which are able to cleave a DNA target sequence from a XP gene can be used for repairing the mutations associated with Xeroderma pigmentosum. Other potential applications include genome engineering at the XP genes loci.
The invention relates to an I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 26 to 40 and 44 to 77 of I-CreI, and is able to cleave a DNA target sequence from a xeroderma pigmentosum (XP) gene.
The cleavage activity of the variant according to the invention may be measured by any well-known, in vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736 or in Arnould et al., J. Mol. Biol., 2006, 355: 443-458. For example, the cleavage activity of the variant of the invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic DNA target sequence within the intervening sequence, cloned in a yeast or a mammalian expression vector. Expression of the variant results in a functional endonuclease which is able to cleave the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene, whose expression can be monitored by appropriate assay.
DEFINITIONS




Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, in which, for example, Q means Gln or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.




Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.
by “I-CreI” is intended the wild-type I-CreI having the sequence SWISSPROT P05725, corresponding to SEQ ID NO: 217 in the sequence listing, or pdb accession code 1g9y. by “I-CreI variant” or “variant” is intended a protein obtained by replacement of at least one amino acid of I-CreI with a different amino acid. by “functional I-CreI variant” is intended a I-CreI variant which is able to cleave a DNA target, preferably a DNA target which is not cleaved by I-CreI. For example, such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. by “I-CreI variant with novel specificity” is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease. The terms “novel specificity”, “modified specificity”, “novel cleavage specificity”, “novel substrate specificity” which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence. by “I-CreI site” is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by I-CreI. I-CreI sites include the wild-type (natural) non-palindromic I-CreI homing site and the derived palindromic sequences such as the sequence 5′-t −12 c −11 a −10 a −9 a −8 a −7 c −6 g −5 t −4 c −3 g −2 t −1 a +1 c +2 g +3 a +4 c +5 g +6 t +7 t +8 t +9 t +10 g +11 a −12 (SEQ ID NO:25), also called C1221 ( FIGS. 3 and 9 ). by “domain” or “core domain” is intended the “LAGLIDADG Homing Endonuclease Core Domain” which is the characteristic α 1 β 1 β 2 α 2 β 3 β 4 α 3 fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues. Said domain comprises four beta-strands (β 1 , β 2 , β 3 , β 4 ) folded in an antiparallel beta-sheet which interacts with one half of the DNA target. This domain is able to associate with another LAGLIDADG Homing Endonuclease Core Domain which interacts with the other half of the DNA target to form a functional endonuclease able to cleave said DNA target. For example, in the case of the dimeric homing endonuclease I-CreI (163 amino acids), the LAGLIDADG Homing Endonuclease Core Domain corresponds to the residues 6 to 94. by “subdomain” is intended the region of a LAGLIDADG Homing Endonuclease Core Domain which interacts with a distinct part of a homing endo-nuclease DNA target half-site. Two different subdomains behave independently and the mutation in one subdomain does not alter the binding and cleavage properties of the other subdomain. Therefore, two subdomains bind distinct part of a homing endonuclease DNA target half-site. by “beta-hairpin” is intended two consecutive beta-strands of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease core domain (β 1 β 2 or, β 3 β 4 ) which are connected by a loop or a turn, by “single-chain meganuclease”, “single-chain chimeric meganuclease”, “single-chain meganuclease derivative”, “single-chain chimeric meganuclease derivative” or “single-chain derivative”, is intended a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic spacer. The single-chain meganuclease is able to cleave a chimeric DNA target sequence comprising one different half of each parent meganuclease target sequence. by “DNA target”, “DNA target sequence”, “target sequence”, “target-site”, “target”, “site”; “site of interest”; “recognition site”, “recognition sequence”, “homing recognition site”, “homing site”, “cleavage site” is intended a 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease. These terms refer to a distinct DNA location, preferably a genomic location, at which a double stranded break (cleavage) is to be induced by the endonuclease. The DNA target is defined by the 5′ to 3′ sequence of one strand of the double-stranded polynucleotide, as indicated above for C1221. Cleavage of the DNA target occurs at the nucleotides in positions +2 and −2, respectively for the sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an I-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target. by “DNA target half-site”, “half cleavage site” or half-site” is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain. by “chimeric DNA target” or “hybrid DNA target” is intended the fusion of a different half of two parent meganucleases target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target). by “XP gene” is intended a gene of one the xeroderma pigmentosum complementation groups (XP-A, XP-B, XP-C, XP-D, XP-E, XP-f) of a mammal. For example, the human XP genes are available in the NCBI database, under the indicated accession numbers: XPA: GeneID:7507, ACCESSION NC — 000009, REGION: complement (97516747 . . . 97539194); XPB: GeneID:2071, ACCESSION NC — 000002, REGION: complement (127731096 . . . 127767982); XPC: GeneID:7508, ACCESSION NC — 000003, REGION: complement (14161651 . . . 14195087); XPD: GeneID:2068, ACCESSION NC — 000019, REGION: complement(50546686 . . . 50565669); XPE: GeneID:1642, ACCESSION NC — 000011, REGION: complement (60823502 . . . 60857125); XPF: GeneID:2072, ACCESSION NC — 000016, REGION: 13921524 . . . 13949705; XPG: GeneID:2073, ACCESSION NC — 000013, REGION: 102296421 . . . 102326346. by “DNA target sequence from a XP gene” “genomic DNA target sequence”, “genomic DNA cleavage site”, “genomic DNA target” or “genomic target” is intended a 20 to 24 bp sequence of a XP gene of a mammal which is recognized and cleaved by a meganuclease variant or a single-chain chimeric meganuclease derivative. by “vector” is intended a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. by “homologous” is intended a sequence with enough identity to another one to lead to a homologous recombination between sequences, more particularly having at least 95% identity, preferably 97% identity and more preferably 99%. “Identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. “individual” includes mammals, as well as other vertebrates (e.g., birds, fish and reptiles). The terms “mammal” and “mammalian”, as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include humans and other primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and ruminants (e.g., cows, pigs, horses). by mutation is intended the substitution, deletion, addition of one or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence. Said mutation can affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

According to the present invention, the positions of the mutations are indicated by reference to the I-CreI amino acid sequence SEQ ID NO: 217.
In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of I-CreI are in positions 44, 68, 70, 75 and/or 77.
In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 26 to 40 of I-CreI are in positions 28, 30, 32, 33, 38 and/or 40.
In another preferred embodiment of said variant, said substitution(s) are in the subdomains situated from positions 28 to 40 and 44 to 70 of I-CreI, preferably in positions 28, 30, 32, 33, 38, 44, 68 and/or 70.
In another preferred embodiment of said variant, it comprises the substitution of the aspartic acid in position 75 by an uncharged amino acid, preferably an asparagine (D75N) or a valine (D75V).
In another preferred embodiment of said variant, it comprises one or more substitutions at additional positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. The I-CreI interacting residues are well-known in the art. The residues which are mutated may interact with the DNA backbone or with the nucleotide bases, directly or via water molecule.
In another preferred embodiment of said variant, it comprises one or more additional mutations that improve the binding and/or the cleavage properties of the variant towards the DNA target sequence of the XP gene. The additional residues which are mutated may be on the entire I-CreI sequence. These mutations may be substitutions in positions 19, 24, 42, 69, 80, 85, 87, 87, 109, 133 and 161. These mutations may affect the active site (position 19), the protein-DNA interface (for example, position 69), the hydrophobic core (for example, positions 85, 87 or 109) or the C-terminal part (for example, position 161).
In yet another preferred embodiment of said variant, said substitutions are replacement of the initial amino acids with amino acids selected from the group consisting of: A, D, E, G, H, K, N, P, Q, R, S, T, Y, C, W, L and V.
The variant according to the present invention may be an homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence. Alternatively, said variant is an heterodimer, resulting from the association of a first and a second monomer having different mutations in positions 26 to 40 and 44 to 77 of I-CreI, preferably in positions 28 to 40 and 44 to 70, said heterodimer being able to cleave a non-palindromic DNA target sequence from a XP gene.
The DNA target sequence which is cleaved by said variant may be in an exon or in an intron of the XP gene. Preferably, it is located, either in the vicinity of a mutation, preferably within 500 bp of the mutation, or upstream of a mutation, preferably upstream of all the mutations of said XP gene.
In another preferred embodiment of said variant, said DNA target sequence is from a human XP gene (XPA to XPG genes).
DNA targets from each human XP gene are presented in Tables IX to XV and FIGS. 16 to 22 .
For example, the sequences SEQ ID NO: 1 to 24 are DNA targets from the XPC gene; SEQ ID NO: 1 to 23 are situated in or close to one of the exons and these sequences cover all the exons of the XP gene (Table XI and FIG. 18 ). The target sequence SEQ ID NO: 24 (Xa.1) is situated in the third intron, upstream of the mutations ( FIG. 1A ). The target sequence SEQ ID NO: 12 (Xc.1) is situated in Exon 9, in the vicinity of the deletion 1132AA and the insertion insVAL580 ( FIG. 1A ).
Hererodimeric variants which cleave each DNA target are presented in Tables I to VIII and FIGS. 16 to 22 .
The sequence of each variant is defined by its amino acid residues at the indicated positions. For example, the first heterodimeric variant of Table I consists of a first monomer having K, S, R, D, K, R, G and N in positions 28, 33, 38, 40, 44, 68, 70 and 75, respectively and a second monomer having R, D, R, K, A, S, N and I in positions 28, 30, 38, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-CreI sequence SWISSPROT P05725, SEQ ID NO: 217 or pdb accession code 1 g9y; I-CreI has G, I, Q, K, N, S, Y, Q, S, A, Q, R, D, R, D, I, E, H, F, I, A and S, in positions 19, 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 68, 69, 70, 75, 77, 80, 85, 87, 109, 133 and 161, respectively. The variant may consist of an I-CreI sequence having the amino acid residues as indicated in the Table. In this case, the positions which are not indicated are not mutated and thus correspond to the wild-type I-CreI sequence. Alternatively, the variant may comprise an I-CreI sequence having the amino acid residues as indicated in the Table. In the latter case, the positions which are not indicated may comprise mutations as defined above, or may not be mutated. For example, the variant may be derived from an I-CreI scaffold protein encoded by SEQ ID NO: 26, said I-CreI scaffold protein (SEQ ID NO: 218) having the insertion of an alanine in position 2, the substitutions A42T, D75N, W110E and R111Q and three additional amino acids (A, A and D) at the C-terminus. In addition, said variant, derived from wild-type I-CreI or an I-CreI scaffold protein, may comprise additional mutations, as defined above.
The target which is cleaved by each heterodimeric variant is indicated in the last column of the Table.





TABLE I



Sequence of heterodimeric I-CreI variants having a DNA

target site in or close to one exon of the XPA gene







Exon closest to the



target sequence

First monomer
Second monomer
(SEQ ID NO: 45 to 57)



28K33S38R40D44K68R70G75N
28R30D38R44K68A70S75N77I
Exon 1

28K30G38H44Q68R70Q75N
28K30G38K44Q68R70S75R77T80K
Exon 1

28K33T38A40Q44N68K70S75R77N
30D33R38G44N68K70S75R77N
Exon 2

28K30G38H44R68Y70S75E77Y
28K33N38Q40Q44K68R70E75N
Exon 3

28K30N38Q44Q68A70N75N
28Q33Y38R40K42R44Q70S75N77N
Exon 4

30N33H38Q44K68R70E75N
28K33R38A40Q44Q68R70S75N
Exon 4

28K30N38Q44K68H70E75N
28K33R38A40Q44Q68R70S75N
Exon 5

28K30G38G44Q68R70G75N
30D33R38G44Q68R70S75N
Exon 5

28K30G38H44Q68R70S75R77T80K
28K33T38A40Q44Q68R70G75N
Exon 6

30N33H38Q44Q68A70N75N
24I26Q28K30N33Y38Q40S44K68R70E75N
Exon 6

28K30N38Q44K68A70N75N
28K33R38A40Q44A68R70G75N
Exon 6

28K33R38E40R44K68S70N75N
28K30G38H44R68Y70S75E77Y
Exon 6

28K33R38A40Q44N68R70N75N
30D33R38G44A68N70N75N
Exon 6









































TABLE II



Sequence of heterodimeric I-CreI variants having

a DNA target site in or close to one exon of the XPB gene







Exon



closest



to the



target



sequence



(SEQ ID



NO: 58 to

First monomer
Second monomer
86)



30R33G38S44K68S70N75N
30N33H38Q44Q68R70S75R77T80K
exon 1

30N33H38A44K68R70E75N
30D33R38T44K68S70N75N
exon 2

28K33N38Q40Q44R68R70R75N
28K33R38A40Q44R68R70R75N
exon 3

28R30D38Q44E68R70A75N
28Q33S38R40K44K68T70T75N
exon 3

30N33H38A44A68R70G75N
28K33T38A40Q68K44Q68Y70S75R77Q
exon 3

30N33H38A44A68N70N75N
30N33H38Q44R68Y70S75E77Y
exon 4

28K33S38Q40Q44R68R70R75N
28K33R38Q40S44K68R70G75N
exon 5

28K33T38A40A44Q68R70S75R77T80K
30N33H38Q44K68Y70S75Q77N
exon 6

30N33T38A42R44Q70S75N77N
28R33A38Y40Q44K68R70E75N
exon 7

30D33R38G44K68A70N75N
28K30G38H44R68R70R75N
exon 7

28R33A38Y40Q44Q68R70G75N
30N33H38A44A68S70R75N
exon 7

28Q33Y38Q40K44A68N70N75N
30D33R38G44Q68R70S75R77T80K
exon 8

30N33H38Q44K68Y70S75D77T
28Q33Y38Q40K44K68Y70S75D77T
exon 9

28K33S38Q40Q44Q68R70S75N
28K30G38H44K68H70E75N
exon 9

30N33H38A44A68R70S75N
28K30G38H44A68N70N75N
exon 9

30D33R38T44K68S70N75N
30D33R38G44R68Y70S75E77Y
exon 10

24I28Q28K30N33Y38Q40S44K68H70E75N
28K30N38Q44K68Y70S75D77T
exon 10

28K33T38A40A44K68Y70S75Q77N
30N33H38Q44Q68R70G75N
exon 10

30N33H38A44N68K70S75R77N
28R33A38Y40Q44R68Y70S75E77Y
exon 11

28Q33Y38Q40K44K68T70T75N
24I26Q28K30N33Y38Q40S44E68R70A75N
exon 11

24I28Q28K30N33Y38Q40S44Q68R70N75N
30D33R38T44A68R70S75Y77Y
exon 12

28K33T38A40Q44Q68R70Q75N
28K30N38Q44Q68Y70S75R77Q
exon 13

28K33S38R40D44Y68D70S75R77T
28K33S38R40D44K68T70T75N
exon 13

30N33H38A44R68Y70S75E77Y
28R33A38Y40Q44Q68R70S75R77T80K
exon 14

28Q33Y38R40K44Q68R70S75R77T80K
30R33G38S44R68R70R75N
exon 14

30R33G38S44Q68R70S75N
28R30D38Q44K68A70N75N
exon 14

30D33R38T42R44Q70S77N
30N33T38A44A68R70S75N
exon 15

30N33T38A44Q68R70S75N
30N33H38Q44E68R70A75N
exon 15

30N33H38A44K68A70N75N
30N33T38Q44A68S70R75N
exon 15






























































TABLE III



Sequence of heterodimeric I-CreI variants having

a DNA target site in or close to one exon of the XPC gene







Exon



closest to



the target



sequence



(SEQ ID

First monomer
Second monomer
NO: 1 to 23)



30D33R38G44R68Y70S75Y77T
28K33R38E40R44R68Y70S75E77V
exon 1

30D33R38T44T68Y70S75R77T
28K33R38N40Q44N68R70N75N
exon 2

28K33R38E40R44R68Y70S75E77I
28Q33Y38R40K42R44Q70S77N
exon 3

28Q33S38R40K44Q68Y70S75N77Y
28T33T38Q40R44T68E70S75R77R
exon 4

30D33R38T44N68R70S75Q77R
28R33A38Y40Q44D68Y70S75S77R
exon 4

28K33R38Q40A44T68R70S75Y77T133V
28K33R38E40R44K68Y70S75D77T
exon 5

28K33N38Q40Q44R68Y70S75E77I
28K33T38A40Q44K68Q70S75N77R
exon 6

28E33R38R40K44Q68Y70S75N77Y
28Q33S38R40K44A68R70S75R77L
exon 7

28K33T38A40Q44A68R70S75R77L
28A33T38Q40R44R68S70S75E77R
exon 8

28K30N38Q44Q68R70S75R77T80K
28T33T38Q40R44T68Y70S75R77V
exon 9

28K30N38Q44A68Y70S75Y77K
28K33R38E40R44T68R70S75Y77T133V
exon 9

28K33R38Q40A44Q68R70N75N
28K33R38A40Q44Q68R70S75N77K
exon 9

33H75N
33R38A40Q44K70N75N
exon 9

30N33H38Q44K68A70S75N77I
28K33N38Q40Q44R68Y70S75E77V
exon 9

28Q33S38R40K44N68R70S75R77D
28T33R38Q40R44Q68R70S75R77T80K
exon 10

28Q33R38R40K44T68Y70S75R77T
28Q33Y38R40K44Y68D70S75R77V
exon 10

28K33T38A40A44T68E70S75R77R
28K30N38Q44A68N70S75Y77R
exon 10

28Q33Y38Q40K44Q68R70S75R77T80K
28K33R38Q40A44Q68R70N75N
exon 10

28K33R38A40Q44K68A70S75N77I
28K33R38E40R44R68Y70S75Y77T
exon 11

28T33T38Q40R44Q68R70S75D77K
28E33R38R40K44Q68R70S75R77T80K
exon 12

28R33A38Y40Q44Q68R70S75R77T80K
28Q33Y38R40K44T68Y70S75R77T
exon 13

28Q33S38R40K42T44K70S75N77Y
28R33A38Y40Q44A68R70S75E77R
exon 14

30D33R38T44A68Y70S75Y77K
28Q33Y38R40K42R44Q70S75N77N
exon 15

28K33R38E40R44K68Q70S75N77R
28Q33S38R40K44R68Y70S75E77V
exon 16























































TABLE IV



Sequence of heterodimeric I-CreI variants having

a DNA target site in or close to one exon of the XPD gene







Exon closest



to the target



sequence



(SEQ ID NO:

First monomer
Second monomer
87 to 119)



30A33D38H44K68R70E75N
28K30G38K44K68S70N75N
exon 1

30N33T38Q44Q68R70G75N
30N33H38Q44A68R70S75E77R
exon 2

30N33H38A44N68R70N75N
30N33H38Q44Q68R70S75R77T80K
exon 3

28K33R38E40R44E68R70A75N
28R33A38Y40Q44A68S70R75N
exon 3

28K33T38R40Q44K68R70E75N
28Q33S38R40K44K68R70G75N
exon 4

30D33R38T44K68R70E75N
28K30G38G44Q68Y70S75R77Q
exon 5

30D33R38T44K68R70E75N
30R33G38S44A68R70S75N
exon 5

28K30G38H44A68R70N75N
28K33S38R40D44A68N70N75N
exon 6

30N33H38Q44Y68D70S75R77T
28R30D38R44A68N70N75N
exon 7

28K33R38Q40S44K68T70T75N
30D33R38G44K68R70E75N
exon 7

28R33A38Y40Q44Q68R70Q75N
28K30G38H44R68Y70S75E77Y
exon 8

33R38E44Q68R70G75N
28K33R38E40R44K68R70E75N
exon 8

30R33G38S44K68R70E75N
30D33R38G44A68R70N75N
exon 9

30R33G38S44A68R70S75N
30D33R38G44K68R70G75N
exon 10

30N33H38A44K68R70G75N
28Q33Y38R40K44K68A70N75N
exon 10

30N33T38A44N68R70N75N
28K30G38K44Q68R70S75N
exon 11

28R33A38Y40Q44K68R70G75N
28Q33Y38R40K44T68Y70S75R77V
exon 12

28Q33Y38R40K44K68Y70S75Q77N
33T44Q68R70S75N
exon 12

28K30G38G44A68N70N75N
28K33R38E40R44E68R70A75N
exon 13

28K33R38A40Q44R68R70R75N
28K33R38E40R44T68Y70S75R77V
exon 14

28K33R38Q40S44K68R70E75N
30R33G38S44A68R70S75E77R
exon 15

28Q33Y38R40K44D68R70N75N
28K33R38A40Q44R68R70R75N
exon 16

30N33H38A44Q68R70G75N
28Q33Y38R40K44A68S70R75N
exon 16

30D33R38T44A68R70N75N
28K33R38E40R44A68R70S75N
exon 17

28Q33Y38R40K44E68R70A75N
33R38E44N68R70N75N
exon 17

28K30G38H44A68R70S75N
30N33T38Q44R68Y70S75E77Y
exon 17

28K33R38E40R44Q68R70G75N
30N33T38A44R68R70R75N
exon 18

28Q33Y38Q40K44Q68R70S75R77T80K
30R33G38S44G68Q70T75N
exon 19

30N33H38Q44D68R70N75N
28K30G38K44G68Q70T75N
exon 19

28K33N38Q40Q44A68N70N75N
28K30G38H44Q68R70G75N
exon 20

28K33R38E40R44K68R70E75N
28K30G38H44A68R70S75N
exon 22

28Q33S38R40K44K68R70E75N
28K33T38A40A44A68R70S75N
exon 22

28K33N38Q40Q44K68T70T75N
28K30G38H44Q68A70N75N
exon 23































































TABLE V



Sequence of heterodimeric I-CreI variants having a DNA target site in

or close to one exon of the XPE gene







Exon closest to the target

First monomer
Second monomer
sequence (SEQ ID NO: 120 to 166)



28K30N38Q44K68R70E75N
28Q33S38R40K44A68S70R75N
exon 1

28K30G38H44Q68R70N75N
28K30G38K44Y68D70S75R77T
exon 2

28K30N38Q44D68Y70S75S77R
28R33A38Y40Q44R68Y70S75E77Y
exon 3

30N33H38Q44Q68R70S75N
30N33H38A44A68R70S75N
exon 4

28Q33S38R40K44K68T70G75N
28K33R38Q40S44A68R70S75E77R
exon 4

28K30G38H44K68R70E75N
30N33H38A44K68R70G75N
exon 5

28R33A38Y40Q44D68R70R75N
28K33N38Q40Q44Q68R70S75R77T80K
exon 5

28K33R38E40R44R68R70R75N
30Q33G38H44A68R70G75N
exon 6

30D33R38T44K68R70E75N
28Q33Y38R40K44A68R70S75N
exon 6

44Q68R70Q75N
33T44Q68Y70S75R77Q
exon 7

28K33T38R40Q44A68R70G75N
28K33R38E40R44Q68R70S75R77T80K
exon 8

30D33R38G44K68S70N75N
30D33R38T44A68S70R75N
exon 8

28Q33Y38Q40K44K68R70E75N
28K30G38H44A68S70R75N
exon 9

30N33H38A44D68Y70S75S77R
28K33R38E40R44Q68R70S75R77T80K
exon 9

28K33S38Q40Q44T68R70S75Y77T133V
30N33T38Q44Q68R70S75N
exon 10

30D33R38T44K68R70E75N
28Q33Y38R40K44A68S70R75N
exon 11

30D33R38T44Q68R70S75N
28K33R38Q40S44K68Y70S75D77T
exon 12

28R33A38Y40Q44K68T70G75N
44N68R70R75N
exon 13

28K33R38E40R44Q68R70G75N
28K33T38R40Q44Q68Y70S75R77Q
exon 13

30N33H38Q44R68Y70S75E77Y
28K33R38Q40S70S75N
exon 14

28R33A38Y40Q44T68Y70S75R77V
30R33G38S44A68R70S75Y77Y
exon 15

28K33R38A40Q44K68R70E75N
28K30G38H44N68R70R75N
exon 16

28K33R38Q40S44D68R70N75N
28K33S38R40D44A68R70G75N
exon 16

30D33R38G44K68H70E75N
28K30G38G44A68R70G75N
exon 17

30N33H38Q44R68R70R75N
30N33T38A44K68R70E75N
exon 17

30D33R38T44K68T70G75N
30R33G38S44Q68R70N75N
exon 18

28K30G38H44R68Y70S75E77Y
28R33A38Y40Q44Q68R70S75R77T80K
exon 19

28R33A38Y40Q44A68R70S75N
28K30G38H44Q68R70S75R77T80K
exon 19

30N33H38A44K68A70N75N
28Q33Y38Q40K44Y68D70S75R77T
exon 20

28Q33Y38R40K44K68R70E75N
28K33R38E40R44N68R70R75N
exon 20

28K33R38A40Q44K68Y70S75Q77N
28K33S38Q40Q44K68R70E75N
exon 21

28Q33Y38R40K44D68R70R75N
30N33H38A44A68R70N75N
exon 21

30D33R38G44A68N70N75N
30D33R38T44K68R70E75N
exon 22

28K33N38Q40Q44D68R70N75N
30D33R38T44A68R70S75N
exon 23

30N33H38A44Q68R70S75N
30Q33G38H44A68N70N75N
exon 24

28R33A38Y40Q44K68R70E75N
28K33R38Q40S44E68R70A75N
exon 24

28R30D38Q44K68R70E75N
28K33S38R40D70S75N
exon 25

30A33D38H44K68H70E75N
28K33T38A40A44N68R70R75N
exon 25

28K30N38Q44K68A70S75N77I
28K33R38E40R44A68S70R75N
exon 26

28Q33Y38Q40K44K68R70E75N
28Q33Y38Q40K44Q68R70S75R77T80K
exon 27

30D33R38G44N68R70A75N
28Q33Y38Q40K44Q68R70S75R77T80K
exon 27

30N33H38Q44R68R70R75N
28R33A38Y40Q44A68R70S75N
exon 27

28K33S38Q40Q44R68Y70S75E77I
30N33T38A44A68R70S75N
exon 27

28K30G38K44N68R70N75N
28K33R38E40R44D68Y70S75S77R
exon 27

28K33R38A40Q44K68R70E75N
28K30N38Q44Q68Y70S75R77Q
exon 27

28K33T38R40Q44K68R70G75N
28R33A38Y40Q44E68R70A75N
exon 27

28K33N38Q40Q44Q68Y70S75R77Q
30D33R38T44Q68R70G75N
exon 27










































































TABLE VI



Sequence of heterodimeric I-CreI variants having

a DNA target site in or close to one exon of the XPF gene







Exon closest



to the target



sequence



(SEQ ID NO:

First monomer
Second monomer
167 to 188)



30N33H38Q44T68Y70S75R77V
30D33R38T44Q68R70G75N
exon 1

28K33S38R40A44K68S70N75N
28E33R38R40K44Q68R70G75N
exon 2

28K33T38A40A44Q68R70N75N
30D33R38G44Q68R70S75N
exon 3

28Q33S38R40K44R68Y70S75E77Y
28K33T38A40Q44T68Y70S75R77T
exon 3

28R30D38Q44Q68R70G75N
28R33A38Y40Q44Q68R70S75R77T80K
exon 4

28K30N38Q44A68R70S75Y77Y
28K33T38R40Q44A68R70S75Y77Y
exon 5

28K33T38R40Q44Q68Y70S75R77Q
28K30N38Q44K68Y70S75Q77N
exon 5

28K33T38A40Q44Q68R70S75N
28R30D38Q44A68N70S75Y77R
exon 6

28K33S38Q40Q44Q68R70S75N
28K33N38Q40Q44A68R70S75Y77Y
exon 6

28K33R38A40Q44E68R70A75N
28K33R38A40Q44Q68R70S75R77T80K
exon 6

28K33T38A40A42T44K70S75N77Y
28K33T38A40A44T68Y70S75R77V
exon 7

30N33T38Q44K68R70E75N
28T33R38Q40R44Q68R70N75N
exon 7

28Q33Y38Q40K44Q68R70G75N
28R30D38Q44T68Y70S75R77V
exon 8

28K33S38Q40Q44Q68R70S75N
28K30N38Q44K68R70G75N
exon 8

28K33T38A40Q44Q68R70G75N
28Q33Y38R40K44Q68R70R75E77R
exon 8

28Q33Y38R40K44Q68R70S75R77T80K
28A33T38Q40R44Q68R70S75R77T80K
exon 9

30D33R38T44A68R70G75N
28K33R38E40R44Q68R70G75N
exon 9

28Q33S38R40K44R68Y70S75D77N
30D33R38T44K68R70E75N
exon 10

28K30G38G44T68Y70S75R77V
28R33A38Y40Q44K68A70S75N77I
exon 11

28Q33Y38R40K44D68Y70S75S77R
30D33R38T44E68R70A75N
exon 11

30N33T38A44Q68R70G75N
28K30G38H44R68Y70S75E77Y
exon 11

28K30G38H44A68N70N75N
30N33H38A44R68Y70S75E77V
exon 11




















































TABLE VII



Sequence of heterodimeric I-CreI variants having

a DNA target site in or close to one exon of the XPG gene







Exon closest to



the target



sequence



(SEQ ID NO:

First monomer
Second monomer
189 to 216)



30D33R38T44K68R70E75N
30D33R38T44Q68N70R75N
exon 1

30D33R38T44E68R70A75N
28T33T38Q40R44Q68Y70S75R77Q
exon 2

30N33Y38Q44Q68R70S75R77T80K
28K33T38A40A44K68R70E75N
exon 3

28T33R38S40R44A68R70S75N
30N33H38Q44N68R70S75R77D
exon 4

28K30G38H44Q68R70Q75N
28T33R38Q40R44A68R70S75N
exon 5

28K33T38R40Q44N68R70A75N
28T33R38Q40R44K68R70E75N
exon 6

28K30G38H44A68Q70N75N
32T33C44Y68D70S75R77T
exon 6

28R30D38Q44T68R70S75Y77T133V
30N33T38A44Q68R70S75N
exon 7

28Q33Y38Q40K44A68R70N75N
28K30N38Q44Q68R70G75N
exon 7

28K33R38E40R44Q68R70S75N
28K33R38Q40A44K68Q70S75N77R
exon 8

30A33D38H44K68G70T75N
28K33T38A40Q44N68R70S75R77D
exon 8

32T33C44N68R70A75N
28R33A38Y40Q44K68R70E75N
exon 8

28K30G38H44A68R70D75N
30N33H38A44Q68R70S75N
exon 8

30R33G38S44K68T70S75N
28R33A38Y40Q42T44K70S75N77Y
exon 8

28R33S38Y40Q44K68T70S75N
28R33A38Y40Q44N68R70S75R77D
exon 8

30N33H38Q44Q68R70D75N
28Q33S38R40K44K68H70E75N
exon 9

28R33S38Y40Q44Y68E70S75R77V
28K30N38Q44K68R70E75N
exon 9

32T33C44A68R70S75N
28T33T38Q40R44T68Y70S75R77T
exon 9

30N33H38A44D68Y70S75S77R
28K33R38E40R44T68Y70S75R77T
exon 10

28R33A38Y40Q44N68R70N75N
28R33A38Y40Q44Q68Y70S75N77Y
exon 11

30N33Y38Q44Q68S70K75N
30D33R38G44A68N70S75Y77R
exon 12

28K33T38A40Q44E68R70A75N
28R33A38Y40Q44S68Y70S75Y77V
exon 12

28A33T38Q40R44E68R70A75N
28K30G38G44A68N70N75N
exon 13

28K33R38A40Q44N68R70N75N
28K33S38R40D42T44K70S75N77Y
exon 13

32T33C44Q68R70D75N
28K33T38A40A44A68R70N75N
exon 14

32T33C44K68A70S75N
30D33R38G44N68R70N75N
exon 14

28Q33Y38R40K44A68R70S75N
30D33R38T42T44K70S75N77Y
exon 15

30D33R38T44K68G70T75N
30D33R38G44A68D70K75N
exon 15


























































TABLE VIII



Sequence of heterodimeric I-CreI variants having a DNA target site

(SEQ ID NO: 24) situated in the third intron of the XPC gene




First monomer
Second monomer



28K30N33S38R40S70S75N
28E30N33Y38R40K44K68S70S75N

28A30N33S38R40K70S75N
28K30G33Y38R40S44K68R70E75N

19A28A30N33S38R40K70S75N
28E30N33Y38R40K44K68R70E75N85R109T

19A28A30N33Y38R40K70S75N87L
28E30N33Y38R40K44K68R70E75N85R109T161F

19A28A30N33S38R40K69G70S75N
28E30N32R33Y38Q40K44K68R70E75N85R109T


28S30N33Y38R40K44K68S70S75N


28S30N33Y38R40K44K68R70D75N


28S30N33Y38R40K44K68A70S75N


28K30G33Y38H40S44K68R70E75N


28K30G33Y38H40S44K68A70G75N


28K30G33Y38R40S44K68R70E75N


28K30G33Y38R40S44K68T70H75N


28K30G33Y38R40S44K68S70S75N


28K30G33Y38R40S44K68T70S75N



































In addition, the variants of the invention may include one or more residues inserted at the NH 2 terminus and/or COOH terminus of the sequence. For example, a tag (epitope or polyhistidine sequence) is introduced at the NH 2 terminus and/or COOH terminus; said tag is useful for the detection and/or the purification of said variant.
The subject-matter of the present invention is also a single-chain chimeric meganuclease derived from an I-CreI variant as defined above. The single-chain chimeric meganuclease is a fusion protein comprising two I-CreI monomers, two I-CreI core domains (positions 6 to 94 of I-CreI) or a combination of both. Preferably, the two monomers/core domains or the combination of both are connected by a peptidic linker.
The subject-matter of the present invention is also a polynucleotide fragment encoding a variant or a single-chain chimeric meganuclease as defined above; said polynucleotide may encode one monomer of an homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease.
The subject-matter of the present invention is also a recombinant vector for the expression of a variant or a single-chain meganuclease according to the invention. The recombinant vector comprises at least one polynucleotide fragment encoding a variant or a single-chain meganuclease, as defined above. In a preferred embodiment, said vector comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant.
A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic DNA. Preferred vectors are those capable of autonomous repli-cation (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), para-myxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 for S. cerevisiae ; tetracycline, rifampicin or ampicillin resistance in E. coli.
Preferably said vectors are expression vectors, wherein the sequence(s) encoding the variant/single-chain meganuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said variant. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome-binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the poly-peptide is expressed. Preferably, when said variant is an heterodimer, the two poly-nucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-β-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), α-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes.
According to another advantageous embodiment of said vector, it includes a targeting construct comprising sequences sharing homologies with the region surrounding the genomic DNA target cleavage site as defined above.
Alternatively, the vector coding for a I-CreI variant and the vector comprising the targeting construct are different vectors.
In both cases, the targeting construct comprises a sequence to be introduced flanked by sequences sharing homologies with the regions surrounding the genomic DNA cleavage sites of the variant as defined here after.
More preferably, said targeting DNA construct comprises:
a) sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above, and
b) a sequence to be introduced flanked by sequences as in a).
Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be located between the two arms. The sequence to be introduced is preferably a sequence which repairs a mutation in the gene of interest (gene correction or recovery of a functional gene), for the purpose of genome therapy. Alternatively, it can be any other sequence used to alter the chromosomal DNA in some specific way including a sequence used to modify a specific sequence, to attenuate or activate the endogenous gene of interest, to inactivate or delete the endogenous gene of interest or part thereof, to introduce a mutation into a site of interest or to introduce an exogenous gene or part thereof. Such chromosomal DNA alterations are used for genome engineering (animal models).
For correcting the XP gene, cleavage of the gene occurs in the vicinity of the mutation, preferably, within 500 bp of the mutation ( FIG. 1B ). The targeting construct comprises a XP gene fragment which has at least 200 bp of homologous sequence flanking the target site (minimal repair matrix) for repairing the cleavage, and includes the correct sequence of the XP gene for repairing the mutation ( FIG. 1B ). Consequently, the targeting construct for gene correction comprises or consists of the minimal repair matrix; it is preferably from 200 pb to 6000 pb, preferably from 1000 pb to 2000 pb.
For example, the target which is cleaved by each of the variant (Tables I to VIII) and the minimal matrix for repairing the cleavage with each variant are indicated in Tables IX to XV and in FIGS. 16 to 22 .





TABLE IX



XPA gene targets cleaved by I-CreI variants









Exon closest

Target


minimal repair


to the target

SEQ ID

target
matrix









sequence
Exon position
NO:
target sequence
position
start
end












Exon 1
1-237
45
ctgcctgcctcggtgcgggcga
123
34
233




Exon 1
1-237
46
cagactggctcgtgcaagccga
290
201
400



Exon 2
3599-3709
47
tttacttacttttgtaggcatg
3582
3493
3692



Exon 3
7719-7824
48
taggacctgttatggaatttga
7716
7627
7826



Exon 4
10097-10262
49
taatctgttttcagagatgctg
10083
9994
10193



Exon 4
10097-10262
50
tgaaactctacttaaagttaca
10240
10151
10350



Exon 5
12318-12435
51
taagctcaaacctcacagtacg
12602
12513
12712



Exon 5
12318-12435
52
tacgacatttctgaaaagcatg
12620
12531
12730



Exon 6
21771-22448
53
cagaattgcggcgagcagtaag
21768
21679
21878



Exon 6
21771-22448
54
tgacctgttttatagaatttta
21933
21844
22043



Exon 6
21771-22448
55
taatacttcagtaataattatg
22044
21955
22154



Exon 6
21771-22448
56
caccactgtaccccaggttcta
22317
22228
22427



Exon 6
21771-22448
57
tgtaccccaggttctagtcatg
22323
22234
22433


























































TABLE X



XPB gene targets cleaved by I-CreI variants









Exon







closest to




minimal repair

the target
Exon
Target

target
matrix









sequence
Position
SEQ ID NO
target sequence
position
start
end












Exon 1
1-123
58
cgggcctgtgggagcggggtca
49
−40
159




Exon 2
459-664
59
tggtatcttgcagacaagaaga
446
357
556



Exon 3
1331-1567
60
ccaacccatgtgcatgagtaca
1424
1335
1534



Exon 3
1331-1567
61
caacccatgtgcatgagtacaa
1425
1336
1535



Exon 3
1331-1567
62
tggaattatgcagtttattaag
1546
1457
1656



Exon 4
3904-3953
63
tgttctcagaaagcagaggcca
3713
3624
3823



Exon 5
4353-4488
64
ttaaatcatggccggcagctca
4197
4108
4307



Exon 6
4676-4840
65
tttgcctgctgattagatttca
5104
5015
5214



Exon 7
5313-5517
66
ttggacctcttagaagatgtaa
5237
5148
5347



Exon 7
5313-5517
67
tatgacttccggaatgattctg
5373
5284
5483



Exon 7
5313-5517
68
ttccctgcggtaagtggtacca
5509
5420
5619



Exon 8
7160-7474
69
ccataccaggtaagcaggctgg
7466
7377
7576



Exon 9
13546-13730
70
cgaccctcgtccgcgaagatga
13606
13517
13716



Exon 9
13546-13730
71
ttaaattttctgattgggccta
13641
13552
13751



Exon 9
13546-13730
72
tgtgctgaggtagctgggcctg
13722
13633
13832



Exon 10
14802-15004
73
tcttactgtattacaggtctgg
14786
14697
14896



Exon 10
14802-15004
74
caaaaccaagaaacgaatcttg
14849
14760
14959



Exon 10
14802-15004
75
tttgccctaaaggaatatgcca
14970
14881
15080



Exon 11
21216-21312
76
cggacctacgtctcagggggaa
21228
21139
21338



Exon 11
21216-21312
77
ccatcttcatatccaaggtttg
21296
21207
21406



Exon 12
22724-22841
78
taaaccactttagttaggaaga
22885
22796
22995



Exon 13
32831-32849
79
cttactggctatttttatattg
32467
32378
32577



Exon 13
32831-32849
80
ctgcatgaatctctgagggcag
33092
33003
33202



Exon 14
34729-34881
81
tggtctctggaatgctagggag
34570
34481
34680



Exon 14
34729-34881
82
tagtcctgcctggatgggccca
34958
34869
35068



Exon 14
34729-34881
83
tgggatttttttggaaatgttg
34980
34891
35090



Exon 15
36450-36887
84
ccttccctccagcgttggccaa
36673
36584
36783



Exon 15
36450-36887
85
ttggctgtgccttcataggtca
36723
36634
36833



Exon 15
36450-36887
86
tgtgccttcataggtcatctag
36728
36639
36838



























































































TABLE XI



XPC gene targets cleaved by I-CreI variants









Exon







closest to

Target


minimal repair

the target
Exon
SEQ ID

target
matrix









sequence
position
NO
target sequence
position
start
end












Exon 1
1-118
1
ccagacgcggaggcctggtgga
194
105
304




Exon 2
5522-5717
2
tcttacttgtcactggggtcca
5793
5704
5903



Exon 3
8034-8146
3
caccatctgaagagaggggcta
8062
7973
8172



Exon 4
10204-10327
4
ctgtctgggttgtgtgggttaa
9976
9887
10086



Exon 4
10204-10327
5
tctgcctgtgaagccagtggag
10262
10173
10372



Exon 5
11331-11415
6
tgagacatatcttcggagggcg
11352
11263
11462



Exon 6
12999-13156
7
tcaaacctggtgaagtggtaag
13140
13051
13250



Exon 7
13651-13771
8
cgggctggggaaagtaggacag
13521
13432
13631



Exon 8
18754-18843
9
tttaattactttcttaggataa
18708
18619
18818



Exon 9
19692-20575
10
caaaatttcttattctgtttaa
19669
19580
19779



Exon 9
19692-20575
11
caagcccatgacctatgtggtg
20392
20303
20502



Exon 9
19692-20575
12
cgagatgtcacacagaggtacg
20438
20349
20548



Exon 9
19692-20575
13
tgacccgcaagtgccgggttga
20478
20389
20588



Exon 10
22091-22252
14
ctgtctggcctttgcagtttca
22074
21985
22184



Exon 10
22091-22252
15
tggcctttgcagtttcaggcta
22079
21990
22189



Exon 10
22091-22252
16
tttgcccactgccattggctta
22117
22028
22227



Exon 10
22091-22252
17
ccatctatcccgagacagctcg
22191
22102
22301



Exon 11
26171-26252
18
tgtcccgcattcccgcagtggg
26106
26017
26216



Exon 12
29639-29773
19
ctaaccgtgctcggaaagcccg
29655
29566
29765



Exon 13
29856-30025
20
ttcccctgctggccaaatgctg
29815
29726
29925



Exon 14
30586-30679
21
ctgtcttccacaaactggggag
30505
30416
30615



Exon 15
31208-31297
22
tctgctcatcagggagaggctg
31255
31166
31365



Exon 16
32428-33437
23
cccaccactgccacctgtccag
32406
32317
32516















































































TABLE XII



XPD gene targets cleaved by I-CreI variants









Exon







closest to

Target


Minimal

the target
Exon
SEQ ID

target
repair matrix









sequence
Position
NO
target sequence
position
start
end












Exon 1
1-36
87
tcgaccccgctgcacagtccgg
2
−87
112




Exon 2
340-439
88
ctatatatcagctactgtgcca
901
812
1011



Exon 3
1425-1502
89
tggtccccaacatgcagggtca
1408
1319
1518



Exon 3
1425-1502
90
cccaacatgcagggtcatggag
1413
1324
1523



Exon 4
1580-1642
91
ctgaacccgtaaaggcagacaa
1515
1426
1625



Exon 5
1829-1942
92
cctgccccccaactttggagta
1806
1717
1916



Exon 5
1829-1942
93
ccttctccttgcccttagccca
1956
1867
2066



Exon 6
5414-5530
94
caggacaggtagcctggggcag
5562
5473
5672



Exon 7
5618-5734
95
tgacctgaaggccctggggcgg
5674
5585
5784



Exon 7
5618-5734
96
cgatactoagtgaggaggctgg
5726
5637
5836



Exon 8
6025-6148
97
otccccggccccccagatcctg
6009
5920
6119



Exon 8
6025-6148
98
cacaacattggtgaggggggcg
6139
6050
6249



Exon 9
6241-6337
99
tgggaccctggcccctgtctga
6379
6290
6489



Exon 10
6453-6586
100
cgggacgagtaccggcgtctgg
6481
6392
6591



Exon 10
6453-6586
101
cgtgctgcccgacgaagtgctg
6561
6472
6671



Exon 11
6661-6829
102
ctggctccatccgcacggccga
6670
6581
6780



Exon 12
8930-9048
103
ctccccgccagattctgtgctg
8919
8830
9029



Exon 12
8930-9048
104
cagcacctacgccaaaggtaag
9032
8943
9142



Exon 13
12873-12942
105
tactcccaggcagtacggggtg
12750
12661
12860



Exon 14
13029-13098
106
tgtcatcatcacatctggggta
13080
12991
13190



Exon 15
13201-13302
107
tgagatccctcccactgtcccg
13173
13084
13283



Exon 16
14844-14907
108
cagcctgggcaacatggtgaca
14703
14614
14813



Exon 16
14844-14907
109
tggtatgctgccagtggggctg
14906
14817
15016



Exon 17
15721-15842
110
cctgacaggcagcctcagtggg
15617
15528
15727



Exon 17
15721-15842
111
cagcatgtaggaatggggtgta
15967
15878
16077



Exon 17
15721-15842
112
caggctgagaatgcagatataa
16025
15936
16135



Exon 18
17238-17330
113
caccacatctcagatgagccag
17112
17023
17222



Exon 19
17417-17489
114
ccatcctgctgtcagtggcccg
17439
17350
17549



Exon 19
17417-17489
115
tggcccggggcaaagtgtccga
17454
17365
17564



Exon 20
17756-17826
116
ccaactcagacacagcatcctg
17661
17572
17771



Exon 22
18220-18363
117
cccactccccaccctcagcctg
18436
18347
18546



Exon 22
18220-18363
118
ctgtcctcctgccctcagcaaa
18459
18370
18569



Exon 23
18851-18984
119
ccaaattcattcaaacatcctg
18730
18641
18840



































































































TABLE XIII



XPE gene targets cleaved by I-CreI variants









Exon closest




minimal repair


to the target

Target

target
matrix









sequence
Exon position
SEQ ID NO
target sequence
position
start
end












Exon 1
1-170
120
caagcctcgacatgtcgtacaa
99
10
209




Exon 2
1387-1535
121
caggacactttacttcggccga
1384
1295
1494



Exon 3
3004-3120
122
taatattggtttccagggggag
2988
2899
3098



Exon 4
3494-3715
123
tggccttttcaaggttattcca
3577
3488
3687



Exon 4
3494-3715
124
ttgtctaccaggtactggatca
3705
3616
3815



Exon 5
6185-6299
125
caggaccctcaggggcggcacg
6182
6093
6292



Exon 5
6185-6299
126
ttccatggtgatcgcaggttgg
6283
6194
6393



Exon 6
7370-7467
127
cccacccataggaatagtcgaa
7123
7034
7233



Exon 6
7370-7467
128
tcttactctgtcgctcaggctg
7742
7653
7852



Exon 7
8941-9099
129
taaaatggctgggtttggtaaa
8842
8753
8952



Exon 8
9984-10067
130
ctgaattatggctgcagttggg
9870
9781
9980



Exon 8
9984-10067
131
ccatcttgtgaaggtgagaaga
10055
9966
10165



Exon 9
10666-10782
132
ccatctcttttgggtgggtctg
10600
10511
10710



Exon 9
10666-10782
133
tggacctggagaggcaggggca
10754
10665
10864



Exon 10
11381-11483
134
ttaaacatatctgaaagtataa
11623
11534
11733



Exon 11
16511-16586
135
tctgaccctaatcgtgagactg
16528
16439
16638



Exon 12
16685-16793
136
tcttctgtggcaacgtggctca
16756
16667
16866



Exon 13
18592-18770
137
ttccccaccagctgtaggtttg
18357
18268
18467



Exon 13
18592-18770
138
tgccatattcttctttgttcag
18846
18757
18956



Exon 14
18868-19031
139
tggcctctggacggacatctcg
18952
18863
19062



Exon 15
19109-19216
140
ttacacagaattcttagtccca
19268
19179
19378



Exon 16
19372-19579
141
tgtcattctctctgtaggtctg
19355
19266
19465



Exon 16
19372-19579
142
tgagatgggaagtatagtgcag
19685
19596
19795



Exon 17
20994-21089
143
ccagaccaatgaaataagagta
20803
20714
20913



Exon 17
20994-21089
144
tggcaccatcgatgagatccag
21027
20938
21137



Exon 18
21183-21294
145
tctgctaccaggaagtgtccca
21188
21099
21298



Exon 19
22660-22783
146
caggctctgtccagcagtgtaa
22657
22568
22767



Exon 19
22660-22783
147
ctccctaatccaggctgttctg
22821
22732
22931



Exon 20
23118-23282
148
tggtctttcagtattcggatgg
23262
23173
23372



Exon 20
23118-23282
149
cagtattcggatggtaagtggg
23270
23181
23380



Exon 21
24001-24095
150
tgtactctatggtggaatttaa
24043
23954
24153



Exon 21
24001-24095
151
tagcacggtgaggcctggacca
24089
24000
24199



Exon 22
29043-29213
152
ccagcccagtattaggggcaga
29294
29205
29404



Exon 23
29923-30032
153
ccaaatgggctgcgttggcaga
29740
29651
29850



Exon 24
30327-30496
154
tggtcttttccacctgggcgag
30369
30280
30479



Exon 24
30327-30496
155
ttccacccccacacaaggctcg
30444
30355
30554



Exon 25
30719-30821
156
caacctcctgctggacatgcag
30750
30661
30860



Exon 25
30719-30821
157
tcgactcaataaagtcatcaaa
30774
30685
30884



Exon 26
32146-32269
158
caagatgcaggaggtggtggca
32239
32150
32349



Exon 27
32859-33624
159
ccatcttctcattgcagtatga
32842
32753
32952



Exon 27
32859-33624
160
tatgacgatggcagcggtatga
32859
32770
32969



Exon 27
32859-33624
161
cgacctcatcaaggttgtggag
32900
32811
33010



Exon 27
32859-33624
162
ctaactcggatccattagccaa
32925
32836
33035



Exon 27
32859-33624
163
tcggatccattagccaagggca
32930
32841
33040



Exon 27
32859-33624
164
tgtcctctttttatttagattg
33319
33230
33429



Exon 27
32859-33624
165
ctgactgccaagccatgggtag
33458
33369
33568



Exon 27
32859-33624
166
ccaaataaagtagaatataaga
33601
33512
33711






























































































































TABLE XIV



XPF gene targets cleaved by I-CreI variants









Exon







closest to

Target


minimal repair

the target
Exon
SEQ ID

target
matrix









sequence
position
NO
target sequence
position
start
end












Exon 1
1-207
167
tgacacagagaaggatggcagg
333
244
443




Exon 2
1866-2046
168
ctgccctgtattaaatagccta
1820
1731
1930



Exon 3
6396-6591
169
tttgatactggtttttgtcatg
6518
6429
6628



Exon 3
6396-6591
170
ctgtatctgtggccaaggtaaa
6575
6486
6685



Exon 4
7863-8070
171
taacccatcgcttgaagtggaa
8007
7918
8117



Exon 5
10545-10725
172
caagactaaatccttagttcag
10589
10500
10699



Exon 5
10545-10725
173
ttgaataaagtgttaggtttta
10765
10676
10875



Exon 6
11992-12120
174
tttaacttttcgtattaggttg
11974
11885
12084



Exon 6
11992-12120
175
ttaacttffcgtattaggttgg
11975
11886
12085



Exon 6
11992-12120
176
tgtaatgtatgttgaaagtata
12265
12176
12375



Exon 7
14027-14137
177
tttgcttccaaaatctatcaaa
14162
14073
14272



Exon 7
14027-14137
178
ttagctctttaaaagtagttca
14199
14110
14309



Exon 8
14981-15578
179
tcatccatccgcttctgggttg
15425
15336
15535



Exon 8
14981-15578
180
ctaacctttgttcggcagcttg
15520
15431
15630



Exon 8
14981-15578
181
tttaatatccgttacgatgctg
15663
15574
15773



Exon 9
17601-17693
182
tagtcctgctcaggaaggatag
17762
17673
17872



Exon 9
17601-17693
183
cctgctcaggaaggatagggca
17766
17677
17876



Exon 10
24558-24670
184
ttgtccctgaagaaagagaagg
24575
24486
24685



Exon 11
27449-28182
185
cactccagaaatgtgcgtggag
27585
27496
27695



Exon 11
27449-28182
186
cagcactggccattacagcaga
27911
27822
28021



Exon 11
27449-28182
187
ctggccattacagcagattctg
27916
27827
28026



Exon 11
27449-28182
188
cagaattagcagccctgtcaca
28052
27963
28162













































































TABLE XV



XPG gene targets cleaved by I-CreI variants









Exon







closest to

Target


minimal repair

the target
Exon
SEQ ID

target
matrix









sequence
position
NO
target sequence
position
start
end












Exon 1
1-285
189
cgggcctgtgggagcggggtca
247
158
357




Exon 2
6049-6224
190
tggtatcttgcagacaagaaga
6045
5956
6155



Exon 3
7688-7803
191
ccaacccatgtgcatgagtaca
7780
7691
7890



Exon 4
8219-8305
192
caacccatgtgcatgagtacaa
8133
8044
8243



Exon 5
9983-10043
193
tggaattatgcagtttattaag
10066
9977
10176



Exon 6
12206-12349
194
tgttctcagaaagoagaggcca
12290
12201
12400



Exon 6
12206-12349
195
ttaaatcatggccggcagctca
12378
12289
12488



Exon 7
15438-15645
196
tttgcctgctgattagatttca
15319
15230
15429



Exon 7
15438-15645
197
ttggacctcttagaagatgtaa
15892
15803
16002



Exon 8
15961-17034
198
tatgacttccggaatgattctg
16130
16041
16240



Exon 8
15961-17034
199
ttccctgcggtaagtggtacca
16235
16146
16345



Exon 8
15961-17034
200
ccataccaggtaagcaggctgg
16263
16174
16373



Exon 8
15961-17034
201
cgaccctcgtccgcgaagatga
16483
16394
16593



Exon 8
15961-17034
202
ttaaattttctgattgggccta
16622
16533
16732



Exon 8
15961-17034
203
tgtgctgaggtagctgggcctg
16976
16887
17086



Exon 9
19598-19842
204
tcttactgtattacaggtctgg
19576
19487
19686



Exon 9
19598-19842
205
caaaaccaagaaacgaatcttg
19816
19727
19926



Exon 9
19598-19842
206
tttgccctaaaggaatatgcca
19871
19782
19981



Exon 10
20193-20312
207
cggacctacgtctcagggggaa
20296
20207
20406



Exon 11
20563-20776
208
ccatcttcatatccaaggtttg
20589
20500
20699



Exon 12
22044-22188
209
taaaccactttagttaggaaga
21979
21890
22089



Exon 12
22044-22188
210
cttactggctatttttatattg
22206
22117
22316



Exon 13
26129-26329
211
ctgcatgaatctctgagggcag
25965
25876
26075



Exon 13
26129-26329
212
tggtctctggaatgctagggag
26539
26450
26649



Exon 14
27190-27274
213
tagtcctgcctggatgggccoa
26921
26832
27031



Exon 14
27190-27274
214
tgggatttttttggaaatgttg
27347
27258
27457



Exon 15
29238-29926
215
ccttccctccagcgttggccaa
29269
29180
29379



Exon 15
29238-29926
216
ttggctgtgccttcataggtca
29545
29456
29655




















































































For example, for correcting some of the mutations in the XPC gene found in Xeroderma pigmentosum (XP), as indicated in FIG. 1A , the following combinations of variants/targeting constructs may be used:
ARG579TER (Exon 4; Premature Stop Codon):
variant: 28Q,33S,38R,40K,44Q,68Y,70S,75N,77Y (first monomer)/28T,33T,38Q,40R,44T,68E,70S,75R,77R (second monomer), and a targeting construct comprising at least positions 9887 to 10086 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant 30D,33R,38T,44N,68R,70S,75Q,77R (first monomer)/28R,33A,38Y,40Q,44D,68Y,70S,75S,77R (second monomer) and a targeting construct comprising at least positions 10173 to 10372 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 6: Substitution PRO218HIS:
variant: 28K,33N,38Q,40Q,44R,68Y,70S,75E,771 (first monomer)/28K,33T,38A,40Q,44K,68Q,70S,75N,77R (second monomer), and a targeting construct comprising at least positions 13051 to 13250 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 9: deletion DEL1132AA or insertion insVAL580:
*variant: 28K,30N,38Q,44Q,68R,70S,75R,77T,80K (first monomer)/28T,33T,38Q,40R,44T,68Y,70S,75R,77V (second monomer) and a targeting construct comprising at least positions 19580 to 19779 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant: 28K,30N,38Q,44A,68Y,70S,75Y,77K (first monomer)/28K,33R,38E,40R,44T,68R,70S,75Y,77T,133V (second monomer) and a targeting construct comprising at least positions 20303 to 20502 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variants: 28K,33R,38Q,40A,44Q,68R,70N,75N (first monomer)/28K,33R,38A,40Q,44Q,68R,70S,75N,77K (second monomer) or 33H,75N (first monomer) and 33R,38A,40Q,44K,70N,75N (second monomer), and a targeting construct comprising at least positions 20349 to 20548 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant: 30N,33H,38Q,44K,68A,70S,75N,771 (first monomer)/28K,33N,38Q,40Q,44R,68Y,70S,75E,77V (second monomer), and a targeting construct comprising at least positions 20389 to 20588 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 14: Substitution LYS822GLN:
variant: 28Q,33S,38R,40K, 42T,44K,70S,75N,77Y (first monomer)/28R,33A,38Y,40Q,44A,68R,70S,75E,77R (second monomer), and a targeting construct comprising at least positions 30416 to 30615 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Alternatively, for restoring a functional gene ( FIG. 1C ), cleavage of the gene occurs upstream of a mutation, for example at position 9119 (target SEQ ID NO: 24). Preferably said mutation is the first known mutation in the sequence of the gene, so that all the downstream mutations of the gene can be corrected simultaneously. The targeting construct comprises the exons downstream of the cleavage site fused in frame (as in the cDNA) and with a polyadenylation site to stop transcription in 3′. The sequence to be introduced (exon knock-in construct) is flanked by introns or exons sequences surrounding the cleavage site, so as to allow the transcription of the engineered gene (exon knock-in gene) into a mRNA able to code for a functional protein ( FIG. 1C ). For example, when cleavage occurs in an exon, the exon knock-in construct is flanked by sequences upstream and downstream of the cleavage site, from a minimal repair matrix as defined above.
The subject-matter of the present invention is also a composition characterized in that it comprises at least one variant, one single-chain chimeric endonuclease and/or at least one expression vector encoding said variant/single-chain molecule, as defined above.
In a preferred embodiment of said composition, it comprises a targeting DNA construct comprising a sequence which repairs a mutation in the XP gene, flanked by sequences sharing homologies with the genomic DNA cleavage site of said variant, as defined above. The sequence which repairs the mutation is either a fragment of the gene with the correct sequence or an exon knock-in construct, as defined above.
Preferably, said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the variant/single-chain chimeric endonuclease according to the invention.
In the case where two vectors may be used, the subject-matter of the present invention is also products containing an I-CreI variant or single-chain chimeric meganuclease expression vector as defined above and a vector which includes a targeting construct as defined above, as a combined preparation for simultaneous, separate or sequential use in Xeroderma pigmentosum.
The subject-matter of the present invention is also the use of at least one meganuclease variant/single-chain chimeric meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing Xeroderma pigmentosum in an individual in need thereof, said medicament being administrated by any means to said individual.
In this case, the use of the meganuclease (variant/single-chain derivative) comprises at least the step of (a) inducing in somatic tissue(s) of the individual a double stranded cleavage at a site of interest comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into the individual a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA. The targeting DNA is introduced into the individual under conditions appropriate for introduction of the targeting DNA into the site of interest.
According to the present invention, said double-stranded cleavage is induced, either in toto by administration of said meganuclease to an individual, or ex vivo by introduction of said meganuclease into somatic cells (skin cells) removed from an individual and returned into the individual after modification.
The subject-matter of the present invention is also a method for preventing, improving or curing Xeroderma pigmentosum in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means.
The meganuclease (variant/single-chain derivative) can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into somatic cells of an individual, by any convenient mean well-known to those in the art, which is appropriate for the particular cell type, alone or in association with either at least an appropriate vehicle or carrier and/or with the targeting DNA.
According to an advantageous embodiment of the uses according to the invention, the meganuclease (polypeptide) is associated with:
liposomes, polyethyleneimine (PEI); in such a case said association is administered and therefore introduced into somatic target cells. membrane translocating peptides (Bonetta, 2002 , The Scientist, 16, 38; Ford et al, Gene Ther, 2001, 8, 1-4; Wadia & Dowdy, 2002 , Curr Opin Biotechnol, 13, 52-56); in such a case, the sequence of the variant/single-chain derivative is fused with the sequence of a membrane translocating peptide (fusion protein).

According to another advantageous embodiment of the uses according to the invention, the meganuclease (polynucleotide encoding said meganuclease) and/or the targeting DNA is inserted in a vector. Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.
Once in a cell, the meganuclease and if present, the vector comprising targeting DNA and/or nucleic acid encoding a meganuclease are imported or translocated by the cell from the cytoplasm to the site of action in the nucleus.
For purposes of therapy, the meganucleases and a pharmaceutically acceptable excipient are administered in a therapeutically effective amount. Such a combination is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality.
In one embodiment of the uses according to the present invention, the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response. A variety of methods for ameliorating or eliminating deleterious immunological reactions of this sort can be used in accordance with the invention. In a preferred embodiment, the meganuclease is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate meganucleases to polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”) (preferably of 500 to 20,000 daltons average molecular weight (MW)). Conjugation with PEG or PPG, as described by Davis et al., (U.S. Pat. No. 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble endonuclease conjugates with anti-viral activity. Similar methods also using a polyethylene-poly-propylene glycol copolymer are described in Saifer et al. (U.S. Pat. No. 5,006,333).
The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector.
The invention also concerns a non-human transgenic animal or a transgenic plant, characterized in that all or part of their cells are modified by a polynucleotide or a vector as defined above.
As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or an eukaryotic cell, such as an animal, plant or yeast cell.
The subject-matter of the present invention is further the use of a meganuclease (variant or single-chain derivative) as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for genome engineering (animal models generation: knock-in or knock-out), for non-therapeutic purposes.
According to an advantageous embodiment of said use, it is for inducing a double-strand break in the gene of interest, thereby inducing a DNA recombination event, a DNA loss or cell death.
According to the invention, said double-strand break is for: repairing a specific sequence, modifying a specific sequence, restoring a functional gene in place of a mutated one, attenuating or activating an endogenous gene of interest, introducing a mutation into a site of interest, introducing an exogenous gene or a part thereof, inactivating or deleting an endogenous gene or a part thereof, translocating a chromosomal aim, or leaving the DNA unrepaired and degraded.
According to another advantageous embodiment of said use, said variant, polynucleotide(s), vector are associated with a targeting DNA construct as defined above.
In a first embodiment of the use of the meganuclease (variant/single-chain derivative) according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at the genomic locus comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease; 2) providing a targeting DNA construct comprising the sequence to be introduced flanked by sequences sharing homologies to the targeted locus. Said meganuclease variant can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell. This strategy is used to introduce a DNA sequence at the target site, for example to generate knock-in or knock-out animal models or cell lines that can be used for drug testing.
The subject-matter of the present invention is also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example a third round of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel, third generation homing endonucleases.
The different uses of the I-CreI variant and the methods of using said I-CreI variant according to the present invention include also the use of the single-chain chimeric meganuclease derived from said variant, the polynucleotide(s), vector, cell, transgenic plant or non-human transgenic mammal encoding said variant or single-chain chimeric endonuclease, as defined above.
The I-CreI variant according to the invention may be obtained by a method for engineering I-CreI variants able to cleave a genomic DNA target sequence from a gene of interest, for example a mammalian gene, comprising at least the steps of:
(a) constructing a first series of I-CreI variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 26 to 40 of I-CreI, preferably from positions 28 to 40 of I-CreI,
(b) constructing a second series of I-CreI variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of I-CreI, preferably from positions 44 to 70 of I-CreI,
(c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions −10 to −8 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions −10 to −8 of said genomic target and (ii) the nucleotide triplet in positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions −10 to −8 of said genomic target
(d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions −5 to −3 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions −5 to −3 of said genomic target and (ii) the nucleotide triplet in positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions −5 to −3 of said genomic target (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions +8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (ii) the nucleotide triplet in positions −10 to −8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +8 to +10 of said genomic target
(f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions +3 to +5 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +3 to +5 of said genomic target and (ii) the nucleotide triplet in positions −5 to −3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target
(g) combining in a single variant, the mutation(s) in positions 28 to 40 and 44 to 70 of two variants from step (c) and step (d), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet in positions −10 to −8 is identical to the nucleotide triplet which is present in positions −10 to −8 of said genomic target, (ii) the nucleotide triplet in positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions −10 to −8 of said genomic target, (iii) the nucleotide triplet in positions −5 to −3 is identical to the nucleotide triplet which is present in positions −5 to −3 of said genomic target and (iv) the nucleotide triplet in positions +3 to +5 is identi-cal to the reverse complementary sequence of the nucleotide triplet which is present in positions −5 to −3 of said genomic target
(h) combining in a single variant, the mutation(s) in positions 28 to 40 and 44 to 70 of two variants from step (e) and step (f), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet in positions +3 to +5 is identical to the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (ii) the nucleotide triplet in positions −5 to −3 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (iii) the nucleotide triplet in posi-tions+8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (iv) the nucleotide triplet in positions −10 to −8 is identical to the reverse complementary sequence of the nucleo-tide triplet in positions +8 to +10 of said genomic target.
(i) combining the variants obtained in steps (g) and (h) to form heterodimers.
(j) selecting and/or screening the heterodimers from step (i) which are able to cleave said genomic DNA target situated in a mammalian gene.
Steps (a), (b), (g), (h) and (i) may further comprise the introduction of additional mutations in order to improve the binding and/or cleavage properties of the mutants. Additional mutations may be introduced at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. This additional step may be performed by generating a library of variants as described in the International PCT Application WO 2004/067736.
The method for engineering I-CreI variants of the invention advantageously comprise the introduction of random mutations on the whole variant or in a part of the variant, in particular the C-terminal half of the variant (positions 80 to 163) to improve the binding and/or cleavage properties of the mutants towards the DNA target from the gene of interest. The mutagenesis may be performed by generating random mutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art and commercially available. Preferably, the mutagenesis is performed on the entire sequence of one monomer of the heterodimer formed in step (i) or obtained in step (j), advantageously on a pool of monomers, preferably on both monomers of the heterodimer of step (i) or (j).
Preferably, two rounds of selection/screening are performed according to the process illustrated by FIG. 25 . In the first round, one of the monomers of the heterodimer is mutagenised (monomer.4 in FIG. 25 ), co-expressed with the other monomer (monomer.3 in FIG. 25 ) to form heterodimers, and the improved monomers.4 are selected against the target from the gene of interest. In the second round, the other monomer (monomer.3) is mutagenised, co-expressed with the improved monomers.4 to form heterodimers, and selected against the target from the gene of interest to obtain meganucleases with improved activity.
The combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques.
The combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), so as to allow the formation of heterodimers. For example, host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the variant(s), so that heterodimers are formed in the host cells.
The selection and/or screening in steps (c), (d), (e), (f) and/or G) may be performed by using a cleavage assay in vitro or in vivo, as described in the International PCT Application WO 2004/067736 or in Arnould et al., J. Mol. Biol., 2006, 355(3): 443-58.
According to another advantageous embodiment of said method, steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break.
The polynucleotide sequence(s) encoding the variant as defined in the present invention may be prepared by any method known by the man skilled in the art. For example, they are amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.
The recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.
The variant of the invention is produced by expressing the poly-peptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed in a host cell modified by one or two expression vector(s), under conditions suitable for the expression or co-expression of the polypeptides, and the variant is recovered from the host cell culture.
Single-chain chimeric meganucleases able to cleave a DNA target from the gene of interest are derived from the variants according to the invention by methods well-known in the art (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention.



In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the I-CreI meganuclease variants and their uses according to the invention, as well as to the appended drawings in which:
FIG. 1 represents the human XPC gene, and two different strate-gies for restoring a functional gene by meganuclease-induced recombination. A. The XPC gene CDS junctions are indicated; the mutations found in the XP-C complementation group are featured by an arrow. The Xa.1 sequence (position 9119, SEQ ID NO: 24) is found in an intronic sequence. The Xc.1 sequence (position 20438, SEQ ID NO: 12) is found in Exon 9. B. Gene correction. A mutation occurs within a known gene. Upon cleavage by a meganuclease and recombination with a repair matrix the deleterious mutation is corrected. C. Exonic sequences knock-in. A mutation occurs within a known gene. The mutated mRNA transcript is featured below the gene. In the repair matrix, exons located downstream of the cleavage site are fused in frame (as in a cDNA), with a polyadenylation site to stop transcription in 3′. Introns and exons sequences can be used as homologous regions. Exonic sequences knock-in results into an engineered gene, transcribed into a mRNA able to code for a functional protein.
FIG. 2 illustrates the principle of the invention. A: Structure of 1-CreI bound to its target. Experimental data have shown that two independent subdomains (squares) could be identified in the DNA binding domain; each subdomain of the core domain binds a different half of the DNA target. B. One would like to identify smaller independent subdomains (squares), each binding a distinct part of a half DNA target. However, there is no structural or experimental data in favour of this hypothesis.
FIG. 3 represents the map of the base specific interactions of 1-CreI with its DNA target C1221 (SEQ ID NO: 25) (Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-74; Chevalier et al. J. Mol. Biol., 2003, 329, 253-69). The inventors have identified novel I-CreI derived endonucleases able to bind DNA targets modified in regions −−10 to −8 and +8 to +10, or −5 to −3 and +3 to +5. These DNA regions are indicated in grey boxes.
FIG. 4 illustrates the rationale of the combinatorial approach. A. Given the separability of the two DNA binding subdomain (top left), one can combine different I-CreI monomers binding different sequences derived from the I-CreI target sequence (top right and bottom left) to obtain heterodimers or single chain fusion molecules cleaving non-palindromic chimeric targets (bottom right). B. The identifi-cation of smaller independent subunit, i.e., subunit within a single monomer or αββαββα fold (top right and bottom left) would allow for the design of novel chimeric molecules (bottom right), by combination of mutations within the same monomer. Such molecules would cleave palindromic chimeric targets (bottom right). C. The combination of the two former steps would allow a larger combinatorial approach, involving four different subdomains (top right, middle left and right, bottom left) that could be combined in new molecules (bottom right). Thus, the identification of a small number of new cleavers for each subdomain would allow for the design of a very large number of novel endonucleases.
FIG. 5 represents the sequences of the cDNA encoding I-CreI N75 scaffold protein and degenerated primers used for the Ulib4 and Ulib5 libraries construction. A. The scaffold (SEQ ID NO: 26) encodes an I-CreI ORF (SEQ ID NO: 218) including the insertion of an alanine codon in position 2, the A42T, D75N, W110E and R111Q codons substitutions and three additional codons (AAD) at the 3′ end. B. Primers (SEQ ID NO: 27, 28, 29),
FIG. 6 represents the cleavage patterns of the I-CreI variants in positions 28, 30, 33, 38 and/or 40. For each of the 141 I-CreI variants obtained after screening, and defined by residues in position 28, 30, 33, 38, 40, 70 and 75, cleavage was monitored in yeast with the 64 targets derived from the C1221 palindromic target cleaved by I-CreI, by substitution of the nucleotides in positions ±8 to 10.Targets are designated by three letters, corresponding to the nucleotides in position −10, −9 and −8. For example GGG corresponds to the tcgggacgtcgtacgacgtcccga target (SEQ ID NO: 30). Values correspond to the intensity of the cleavage, evaluated by an appropriate software after scanning of the filter. For each protein, observed cleavage (black box) or non observed cleavage (0) is shown for each one of the 64 targets. All the variants are mutated in position 75: D75N.
FIG. 7 represents the cleavage patterns of the I-CreI variants in position 44, 68 and/or 70. For each of the 292 I-CreI variants obtained after screening, and defined by residues in position 44, 68 and 70 (first three columns) cleavage was monitored in yeast with the 64 targets derived from the C1221 palindromic target cleaved by I-CreI, by substitution of the nucleotides in positions ±3 to 5. Targets are designated by three letters, corresponding to the nucleotides in position −5, −4 and −3. For each protein, observed cleavage (1) or non observed cleavage (0) is shown for each one of the 64 targets. All the variants are mutated in position 75: D75N.
FIG. 8 represents the localisation of the mutations in the protein and DNA target, on a I-CreI homodimer bound to its target. The two set of mutations (residues 44, 68 and 70; residues 28, 30, 33, 38 and 40 are shown in black on the monomer on the left. The two sets of mutations are clearly distinct spatially. However, there is no structural evidence for distinct subdomains. Cognate regions in the DNA target site (region −5 to −3; region −10 to −8) are shown in grey on one half site.
FIG. 9 represents the Xa series of targets and close derivatives. C1221 (SEQ ID NO: 25) is one of the I-CreI palindromic target sequences. 10TGC_P, 10AGG_P, 5CCT_P and 5TTT (SEQ ID NO: 31, 32, 33, 34) are close derivatives found to be cleaved by I-CreI mutants. They differ from C1221 by the boxed motives. C1221, 10TGC_P, 10AGG_P, 5CCT_P and 5TTT were first described as 24 bp sequences, but structural data suggest that only the 22 bp are relevant for protein/DNA interaction. However, positions ±12 are indicated in parenthesis.Xa.1 (SEQ ID NO:24) is the DNA sequence located in the human XPC gene at position 9119. In the Xa.2 target (SEQ ID NO:35), the TTGA sequence in the middle of the target is replaced with GTAC, the bases found in C1221. Xa.3 (SEQ ID NO:36) is the palindromic sequence derived from the left part of Xa.2, and Xa.4 (SEQ ID NO:37) is the palindromic sequence derived from the right part of Xa.2. The boxed motives from 10TGC_P, 10AGG_P, 5CCT_P and 5TTT are found in the Xa series of targets.
FIG. 10 represents the pCLS1055 plasmid vector map.
FIG. 11 represents the pCLS10542 plasmid vector map.
FIG. 12 illustrates the cleavage of the Xa.3 target. The figure displays secondary screening of the I-CreI K28, N30, S33, R38, S40, S70 N75 (KNSRSSN) and I-CreI A28, N30, S33, R38, K40, S70 N75 (ANSRKSN) mutants with Xa.1, Xa.2, Xa.3 and Xa.4 targets (SEQ ID NO:24, 35, 36, 37).
FIG. 13 illustrates the cleavage of the Xa.4 target. The figure displays secondary screening of a series of combinatorial mutants among those described in Table XVI with Xa.1, Xa.2, Xa.3 and Xa.4 targets (SEQ ID NO:24, 35, 36, 37).
FIG. 14 represents the pCLS1107 vector map.
FIG. 15 illustrates the cleavage of the Xa.1 and Xa.2 targets. A series of I-CreI N75 mutants cutting Xa.4 are co-expressed with either KNSRSS (a) or ANSRKS (b). Cleavage is tested with the Xa.1, Xa.2, Xa.3 and Xa.4 targets (SEQ ID NO:24, 35, 36, 37). Mutants cleaving Xa.1 are circled.
FIGS. 16 to 22 illustrate the DNA target sequences found in each exon of the human XP genes (XPA to XPG gene) and the corresponding I-CreI variant which is able to cleave said DNA target. The exons closest to the target sequences, and the exons junctions are indicated (columns 1 and 2), the sequence of the DNA target is presented (column 3), with its position (column 4). The minimum repair matrix for repairing the cleavage at the target site is indicated by its first nucleotide (start, column 7) and last nucleotide (end, column 8). The sequence of each variant is defined by its amino acid residues at the indicated positions. For example, the first heterodimeric variant of FIG. 16 consists of a first monomer having K, S, R, D, K, R, G and N in positions 28, 33, 38, 40, 44, 68, 70 and 75, respectively and a second monomer having R, D, R, K, A, S, N and I in positions 28, 30, 38, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-CreI sequence SWISSPROT P05725 or pdb accession code 1 g9y; I-CreI has I, Q, K, N, S, Y, Q, S, A, Q, R, R, D, I, E and A, in positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 68, 70, 75, 77, 80 and 133, respectively. The positions which are not indicated may be mutated or not mutated. In the latter case, the positions which are not indicated correspond to the wild-type I-CreI sequence.
FIG. 23 represents three targets, derivated from C1221: Xa.1, Xb.1 and Xc. 1 (SEQ ID NO: 24, 8, 12), identified in the human XPC gene. Each initial target was transformed in a Xx.2 target (SEQ ID NO: 35, 219, 222) more favourable for I-CreI cleavage, then in two palindromics targets Xx.3 (SEQ ID NO: 36, 220, 223) and Xx.4 (SEQ ID NO: 37, 221, 224). Stars represent potential targets found in the gene. Black squares or vertical lines represent the XPC exons.
FIG. 24 illustrates the strategy for the making and screening of custom-designed Homing Endonucleases. A. General strategy of primary screen. Appropriate I-CreI derivatives with locally altered specificities are identified in the database. Then, a combinatorial approach is used to assemble these mutants by in vivo cloning. Active combinatorial mutants are identified as homodimers using a yeast screening assay, on either Xx.3 either Xx.4 targets. Heterodimers were screened by co-expression against both non palindromic targets: Xx.2 and Xx.1. B. Heterodimer Screening Examples. Each new endonuclease is screened on both non palindromic targets: Xx.2 and Xx.1, differing at positions ±2 and ±1. The screening is performed as described previously (International PCT Application WO 2004/067736; Arnould et al., J. Mol. Biol., 2006, 355, 443-458); blue staining indicates cleavage.
FIG. 25 illustrates the strategy of activity improvement of custom-designed Homing Endonucleases. A pool of 4 monomers active against Xx.4 is mutagenized by error-prone PCR while its counterpart (monomers active against Xx.3) is used to generate yeast expression strain containing the target Xx.1. The mutagenized library is transformed in a second yeast strain and cloned by in vivo cloning. Screening of heterodimer activity is performed by mating of the two yeast strains. The same procedure is then repeated on the I-CreI variants active against the Xx.3 target.
FIG. 26 illustrates the screening and characterisation of improved heterodimers cleaving Xb.1 target. A. Final screen of heterodimers from initials to improve versions. Two forms of I-SceI homing endonuclease are used as a control: I-SceI*, I-SceI variant with poor activity; I-SceI, original I-SceI ORF with strong activity. Ø: yeast strain transformed with empty vector. Initial: representative mutant activity before improvement. Yeast strains containing the improved mutant active either against Xa.3, or against Xa.4 were mated with yeast strain containing the improved mutants and the Xa.1 target. B. Protein sequences of the most active I-CreI variants. I: Protein sequence before activity improvement. M: Protein sequence after random mutagenesis by error-prone PCR.
FIG. 27 illustrates the heterodimer activity in mammalian cells. Heterodimer activity was quantified by co-expression assay as described in materials and methods section. Ø: CHO transfected with empty vector. I-SceI: CHO transfected with I-SceI expressing vector. I: mutants before activity improvement. M: mutants after activity improvement. All the target vectors carry an extra sub-optimal 18 bp I-SceI site with poor cleavage efficiency. It is used as control experiment. The I-SceI activity represents the mean obtained with all target vectors.
FIG. 28 represents the profiling of combinatorial homodimeric mutants. A. The half site of I-CreI C1221 palindromic target is indicated on the bottom line of each box. The individual nucleotide changes tolerated at each position are indicated. The size of the letters is proportional to the activity of the enyme I-CreI D75N and wild-type. B. The half site of Xa.3 and Xa.4 palindromic targets are indicated on the bottom line. The individual nucleotide changes tolerated at each position are indicated for a mutant.3 and a mutant.4, respectively. The size of the letter is proportional to the activity of the mutants.



EXAMPLE 1
Engineering of I-CreI Variants with Modified Specificity in Positions ±8 to ±10
The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity, are described in the International PCT Application WO 2004/067736, Arnould et al., J. Mol. Biol., 2006, 355, 443-458, Epinat et al., N.A.R., 2003, 31, 2952-2962 and Chames et al., Nucleic Acids Res., 2005, 33, e178). These assays result in a functional LacZ reporter gene which can be monitored by standard methods.
A) Material and Methods
a) Construction of the Ulib4, Ulib5 and Lib4 Libraries
I-CreI wt and I-CreI D75N open reading frames were synthesized, as described previously (Epinat et al., N.A.R., 2003, 31, 2952-2962). Mutation D75N was introduced by replacing codon 75 with aac. Three combinatorial libraries (Ulib4, Ulib5 and Lib4) were derived from the I-CreI D75N protein by replacing three different combinations of residues, potentially involved in the interactions with the bases in positions ±8 to 10 of one DNA target half-site. The diversity of the meganuclease libraries was generated by PCR using degenerated primers harboring a unique degenerated codon (coding for 10 or 12 different amino acids), at each of the selected positions.
The three codons at positions N30, Y33 and Q38 (Ulib4 library) or K28, N30 and Q38 (Ulib5 library) were replaced by a degenerated codon VVK (18 codons) coding for 12 different amino acids: A,D,E,G,H,K,N,P,Q,R,S,T). In consequence, the maximal (theoretical) diversity of these protein libraries was 12 3 or 1728. However, in terms of nucleic acids, the diversity was 18 3 or 5832. Fragments carrying combinations of the desired mutations were obtained by PCR, using a pair of degenerated primers (Ulib456for and Ulib4rev; Ulib456for and Ulib5rev, FIG. 5B ) and as DNA template, the D75N open reading frame (ORF), ( FIG. 5A ). The corresponding PCR products were cloned back into the I-CreI N75 ORF in the yeast replicative expression vector pCLS0542 (Epinat et al., precited), carrying a LEU2 auxotrophic marker gene. In this 2 micron-based replicative vector, I-CreI variants are under the control of a galactose inducible promoter.
In Lib4, ordered from BIOMETHODES, an arginine in position 70 was first replaced with a serine (R70S). Then positions 28, 33, 38 and 40 were randomized. The regular amino acids (K28, Y33, Q38 and S40) were replaced with one out of 10 amino acids (A,D,E,K,N,Q,R,S,T,Y). The resulting library has a theoretical complexity of 10000 in terms of proteins.
b) Construction of Target Clones
The C1221 twenty-four bp palindrome (tcaaaacgtcgtacgacgttttga, (SEQ ID NO: 25) is a repeat of the half-site of the nearly palindromic natural I-CreI target (tcaaaacgtcgtgagacagtttgg, SEQ ID NO: 38). C1221 is cleaved as efficiently as the I-CreI natural target in vitro and ex vivo in both yeast and mammalian cells. The 64 palindromic targets were derived from C1221 as follows: 64 pairs of oligonucleotides ((ggcatacaagtttcnnnacgtcgtacgacgtnnngacaatcgtctgtca (SEQ ID NO: 39) and reverse complementary sequences) were ordered form Sigma, annealed and cloned into pGEM-T Easy (PROMEGA) in the same orientation. Next, a 400 bp PvuII fragment was excised and cloned into the yeast vector pFL39-ADH-LACURAZ, also called pCLS0042, and the mammalian vector pcDNA3 derivative (pcDNA3.1-LAACZ), both described previously (Epinat et al., 2003, precited), resulting in 64 yeast reporter vectors (target plasmids).
Alternatively, double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotides, was cloned using the Gateway protocol (INVITROGEN) into yeast and mammalian reporter vectors.
c) Yeast Strains
The library of meganuclease expression variants was transformed into the leu2 mutant haploid yeast strain FYC2-6A: alpha, trp1Δ63, leu2Δ1, his3Δ200. A classical chemical/heat choc protocol that routinely gives us 10 6 independent transformants per μg of DNA derived from (Gietz and Woods, Methods Enzymol., 2002, 350, 87-96), was used for transformation. Individual transformant (Leu + ) clones were individually picked in 96 wells microplates.13824 colonies were picked using a colony picker (QpixII, GENETIX), and grown in 144 microtiter plates.
The 64 target plasmids were transformed using the same protocol, into the haploid yeast strain FYBL2-7B: a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202, resulting in 64 tester strains.
d) Mating of Meganuclease Expressing Clones and Screening in Yeast
Meganuclease expressing clones were mated with each of the 64 target strains, and diploids were tested for beta-galactosidase activity, by using the screening assay illustrated on FIG. 2 of Arnould et al., 2006, precited. I-CreI variant clones as well as yeast reporter strains were stocked in glycerol (20%) and replicated in novel microplates. Mating was performed using a colony gridder (QpixII, GENETIX). Mutants were gridded on nylon filters covering YPD plates, using a high gridding density (about 20 spots/cm 2 ). A second gridding process was performed on the same filters to spot a second layer consisting of 64 different reporter-harboring yeast strains for each variant. Membranes were placed on solid agar YPD rich medium, and incubated at 30° C. for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (2%) as a carbon source (and with G418 for coexpression experiments), and incubated for five days at 37° C., to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37° C., to monitor β-galactosidase activity. After two days of incubation, positive clones were identified by scanning. The β-galactosidase activity of the clones was quantified using appropriate software. The clones showing an activity against at least one target were isolated (first screening). The spotting density was then reduced to 4 spots/cm 2 and each positive clone was tested against the 64 reporter strains in quadruplicate, thereby creating complete profiles (secondary screening).
e) Sequence
The open reading frame (ORF) of positive clones identified during the first and/or secondary screening in yeast was amplified by PCR on yeast colonies using primers: PCR-Gal10-F (gcaactttagtgctgacacatacagg, SEQ ID NO:40) and PCR-Gal10-R (acaaccttgattgcagacttgacc, SEQ ID NO:41) or 5′ggggacaagtttgtacaaaaaaggcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc3′ (SEQ ID NO: 225) and 5′ggggaccactttgtacaagaaagctgggtttagtcggccgccggggaggatttcttcttctcgc 3′ (SEQ ID NO: 226), from PROLIGO. Briefly, yeast colony is picked and resuspended in 100 μl of LGlu liquid medium and cultures overnight. After centrifugation, yeast pellet is resuspended in 10 μl of sterile water and used to perform PCR reaction in a final volume of 50 μl containing 1.5 μl of each specific primers (100 pmol/μl). The PCR conditions were one cycle of denaturation for 10 minutes at 94° C., 35 cycles of denaturation for 30 s at 94° C., annealing for 1 min at 55° C., extension for 1.5 min at 72° C., and a final extension for 5 min. The resulting PCR products were then sequenced.
f) Re-Cloning of Primary Hits
The open reading frames (ORFs) of positive clones identified during the primary screening were recloned using the Gateway protocol (Invitrogen). ORFs were amplified by PCR on yeast colonies, as described in e). PCR products were then cloned in: (i) yeast gateway expression vector harboring a galactose inducible promoter, LEU2 or KanR as selectable marker and a 2 micron origin of replication, (ii) a pET 24d(+) vector from NOVAGEN, and (iii) a CHO gateway expression vector pcDNA6.2 from INVITROGEN. Resulting clones were verified by sequencing (MILLEGEN).
B) Results
I-CreI is a dimeric homing endonuclease that cleaves a 22 bp pseudo-palindromic target. Analysis of I-CreI structure bound to its natural target has shown that in each monomer, eight residues establish direct interactions with seven bases (Jurica et al., 1998, precited). According to these structural data, the bases of the nucleotides in positions +8 to 10 establish specific contacts with I-CreI amino-acids N30, Y33 and Q38 ( FIG. 3 ). Thus, novel proteins with mutations in positions 30, 33 and 38 could display novel cleavage profiles with the 64 targets resulting from substitutions in positions ±8, ±9 and ±10 of a palindromic target cleaved by I-CreI. In addition, mutations might alter the number and positions of the residues involved in direct contact with the DNA bases. More specifically, positions other than 30, 33, 38, but located in the close vicinity on the folded protein, could be involved in the interaction with the same base pairs.
An exhaustive protein library vs. target library approach was undertaken to engineer locally this part of the DNA binding interface. First, the I-CreI scaffold was mutated from D75 to N. The D75N mutation did not affect the protein structure, but decreased the toxicity of I-CreI in overexpression experiments.
Next the Ulib4 library was constructed: residues 30, 33 and 38, were randomized, and the regular amino acids (N30, Y33, and Q38) replaced with one out of 12 amino acids (A,D,E,G,H,K,N,P,Q,R,S,T). The resulting library has a complexity of 1728 in terms of protein (5832 in terms of nucleic acids).
Then, two other libraries were constructed: Ulib5 and Lib4. In Ulib5, residues 28, 30 and 38 were randomized, and the regular amino acids (K28, N30, and Q38) replaced with one out of 12 amino acids (ADEGHKNPQRST). The resulting library has a complexity of 1728 in terms of protein (5832 in terms of nucleic acids). In Lib4, an Arginine in position 70 was first replaced with a Serine. Then, positions 28, 33, 38 and 40 were randomized, and the regular amino acids (8, Y33, Q38 and S40) replaced with one out of 10 amino acids (A,D,E,K,N,Q,R,S,T,Y). The resulting library has a complexity of 10000 in terms of proteins.
In a primary screening experiment, 20000 clones from Ulib4, 10000 clones from Ulib5 and 20000 clones from Lib4 were mated with each one of the 64 tester strains, and diploids were tested for beta-galactosidase activity. All clones displaying cleavage activity with at least one out of the 64 targets were tested in a second round of screening against the 64 targets, in quadriplate, and each cleavage profile was established. Then, meganuclease ORF were amplified from each strain by PCR, and sequenced, and 141 different meganuclease variants were identified.
The 141 validated clones showed very diverse patterns. Some of these new profiles shared some similarity with the wild type scaffold whereas many others were totally different. Results are summarized in FIG. 6 . Homing endonucleases can usually accommodate some degeneracy in their target sequences, and the I-CreI N75 scaffold protein itself cleaves a series of 4 targets, corresponding to the aaa, aac, aag, an aat triplets in positions ±10 to ±8. A strong cleavage activity is observed with aaa, aag and aat, whereas AAC is only faintly cut (and sometimes not observed). Similar pattern is found with other proteins, such as I-CreI K28 N30 D33 Q38 S40 R70N75, I-CreI K28 N30 Y33 Q38 S40 R70N75. With several proteins, such as I-CreI R8N30 N33 Q38 D40 S70 N75 and I-CreI K28 N30 N33 Q38 S40 R70N75, aac is not cut anymore.
However, a lot of proteins display very different patterns. With a few variants, cleavage of a unique sequence is observed. For example, protein I-CreI K28 R30G33 T38 S40 R70N75 is active on the “ggg” target, which was not cleaved by wild type protein, while I-CreI Q28 N30 Y33 Q38 R40 S70 N75 cleaves AAT, one of the targets cleaved by I-CreI N75. Other proteins cleave efficiently a series of different targets: for example, I-CreI N28 N30 S33 R38K40 S70 N75 cleaves ggg, tgg and tgt, CreI K28 N30H33 Q38 S40 R70 N75 cleaves aag, aat, gac, gag, gat, gga, ggc, ggg, and ggt. The number of cleaved sequences ranges from 1 to 10. Altogether, 37 novel targets were cleaved by the mutants, including 34 targets which are not cleaved by I-CreI and 3 targets which are cleaved by I-CreI (aag, aat and aac, FIG. 6 ).
EXAMPLE 2
Strategy for Engineering Novel Meganucleases Cleaving a Target from the XPC Gene
A first series of I-CreI variants having at least one substitution in positions 44, 68 and/or 70 of I-CreI and being able to cleave mutant I-CreI sites having variation in positions ±3 to 5 was identified previously (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). The cleavage pattern of the variants is presented in FIG. 7 .
A second series of I-CreI variants having at least one substitution in positions 28, 30, 33 or 28, 33, 38 and 40 of I-CreI and being able to cleave mutant I-CreI sites having variation in positions ±8 to 10 was identified as described in example 1. The cleavage pattern of the variants is presented in FIG. 6 .
Positions 28, 30, 33, 38 and 40 on one hand, and 44, 68 and 70, on another hand are on a same DNA-binding fold, and there is no structural evidence that they should behave independently. However, the two sets of mutations are clearly on two spatially distinct regions of this fold ( FIG. 8 ) located around different regions of the DNA target. These data suggest that I-CreI comprises two independent functional subunits which could be combined to cleave novel chimeric targets. The chimeric target comprises the nucleotides in positions ±3 to 5 and ±8 to 10 which are bound by each subdomain.
This hypothesis was verified by using targets situated in a gene of interest, the XPC gene. The targets cleaved by the I-CreI variants are 24 bp derivatives of C1221, a palindromic sequence cleaved by I-CreI. However, the structure of I-CreI bound to its DNA target suggests that the two external base pairs of these targets (positions −12 and 12) have no impact on binding and cleavage (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774; Chevalier et al., 2003, J. Mol. Biol., 329, 253-269) and in this study, only positions −11 to 11 were considered. Consequently, the series of targets identified in the XPC gene were defined as 22 bp sequences instead of 24 bp.
Xa.1, Xb.1 and Xc. 1 are 22 bp (non-palindromic) targets located at position 9119, 13521 and 20438, respectively of the human XPC gene ( FIGS. 1A , 9 and 23 ). The meganucleases cleaving Xa.1, Xb.1 or Xc. 1 could be used to correct mutations in the vicinity of the cleavage site ( FIG. 1B ). Since the efficiency of gene correction decreases when the distance to the DSB increases (Elliott et al., Mol Cell Biol, 1998, 18, 93-101), this strategy would be most efficient with mutations located within 500 bp of the cleavage site. For example, meganucleases cleaving Xc. 1 could be used to correct mutations in Exon 9 (deletion DEL1132AA or insertion insVAL580, FIG. 1A ). Alternatively, the same meganucleases could be used to knock-in exonic sequences that would restore a functional XPC gene at the XPC locus ( FIG. 1C ). This strategy could be used for any mutation downstream of the cleavage site.
Xa.1 is partly a patchwork of the 10TGC_P, 10AGG_P, 5TTT_P and 5CCT_P targets ( FIGS. 9 and 23 ) which are cleaved by previously identified meganucleases ( FIGS. 6 and 7 ). Thus, Xa.1 could be cleaved by combinatorial mutants resulting from these previously identified meganucleases.
Xb.1 is partly a patchwork of the 10GGG_P, 10TGT_P, 5GGG_P and 5TAC_P targets ( FIG. 23 ) which are cleaved by previously identified mega-nucleases ( FIGS. 6 and 7 ). Thus, Xb.1 could be cleaved by combinatorial mutants resulting from these previously identified meganucleases.
Xc.1 is partly a patchwork of the 10GAG_P, 10GTA_P and 5TCT_P targets ( FIG. 23 ) which are cleaved by previously identified meganucleases ( FIGS. 6 and 7 ), and 5GTC_P which is the sequence of C1221 cleaved by I-CreI. Thus, Xc.1 could be cleaved by combinatorial mutants resulting from these previously identified meganucleases.
Therefore, to verify this hypothesis, two palindromic targets, corresponding to the left (Xx.3) and right half (Xx.4) sequences of the identified targets (Xx. 1) were produced ( FIGS. 9 and 23 ). These two derived palindromic targets keep the GTAC sequence from the C1221 palindromic I-CreI target at positions −2 to +2 ( FIGS. 9 and 23 ). Since Xx.3 and Xx.4 are palindromic, they should be cleaved by homodimeric proteins. In a first step, proteins able to cleave the Xx.3 and Xx.4 sequences as homodimers were designed (examples 3 and 4), as illustrated in FIG. 24A . In a second step, the proteins obtained in examples 3 and 4 were co-expressed to obtain heterodimers cleaving Xx.2 and Xx.1, for some heterodimers (example 5), as illustrated in FIG. 24A .
EXAMPLE 3
Making of Meganucleases Cleaving Xx.3
This example shows that I-CreI mutants can cut the Xx.3 DNA target sequences derived from the left part of the Xx.2 targets in a palindromic form ( FIGS. 9 and 23 ). Target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix_P; for example, target Xa.3 will be noted also ctgccttttgt_P.
Xa.3 is similar to 5TTT_P in positions ±1, ±2, ±3, ±4, ±5 and ±111 and to 10TGC_P in positions ±1, ±2, ±8, ±9, ±10 and ±11.
Xb.3 is similar to 5GGG_P in positions ±1, ±2, ±3, ±4, ±5 and ±11 and to 10GGG_P in positions ±1, ±2, ± 8 , ± 9 , ±10 and ±11
Xc.3 is similar to C1221 in positions ±1, ±2, ±3, ±4, ±5, ±7 and ±11 and to 10GAG_P in positions ±1, ±2, ±7, ±8, ±9, ±10 and ±11
The wild-type I-CreI is known to be tolerant for nucleotide substitutions at positions ±1, ±7 and ±6 (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269; Jurica et al., Mol. Cell., 1998, 2, 469-476). Thus, it was hypothesized that positions +6 and +7 would have little effect on the binding and cleavage activity.
Mutants able to cleave the 5TTT_P and 5GGG_P targets were previously obtained by mutagenesis on I-CreI N75 at positions 44, 68 and 70 or I-CreI S70 at positions 44, 68, 75 and 77, as described in Arnould et al., J. Mol. Biol., 2006, 355, 443-458 ( FIG. 7 ). Mutants able to cleave the 10TGC_P, 10GGG_P, and 10TGA_P targets were obtained by mutagenesis on I-CreI N75 at positions 28, 30, 38, or 30, 33, 38, on I-CreI S70 N75 at positions 28, 33, 38 and 40 and 70, or on I-CreI D75 or N75 at two positions chosen from 28, 30, 32, 33, 38 and 40 (example 1 and FIG. 6 ). Thus, combining such pairs of mutants would allow for the cleavage of the Xx.3 target.
Some sets of proteins are both mutated at position 70. However, it was hypothesized that two separable functional subdomains exist in I-CreI. That implies that this position has little impact on the specificity in bases 10 to 8 of the target.
Therefore, to check whether combined mutants could cleave the Xa.3 and Xb.3 targets, mutations at positions 44, 68, 70 and/or 75 from proteins cleaving the 5NNN region of the target (5TTT_P and 5GGG_P targets) were combined with the 28, 30, 32, 33, 38 and/or 40 mutations from proteins cleaving the 10NNN region of the targets (10TGC_P and 10GGG_P targets), as illustrated in FIG. 24A .
Xc.3 which is identical to C1221 in positions ±3 to 5 should be cleaved by previously identified mutants cleaving the 10 GAG_P target (no combination of mutations).
A) Material and Methods
a) Construction of Target Vector:
The C1221 derived target was cloned as follows: oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence was ordered from Proligo (as example: 5′ tggcatacaagtttctgccttttgtacaaaaggcagacaatcgtctgtca 3′ (SEQ ID NO: 42, for the Xa.3 target). Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway R protocol (INVITROGEN) into yeast reporter vector (pCLS1055, FIG. 10 ). Yeast reporter vector was transformed into S. cerevisiae strain FYBL2-7B (MAT alpha, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202).
b) Construction of Combinatorial Mutants:
I-CreI mutants cleaving 10TGC_P, 10GGG_P, 5TTT_P or 5GGG_P were identified as described in example 1 and FIG. 6 , and Arnould et al., J. Mol. Biol., 2006, 355, 443-458 and FIG. 7 , respectively for the 10TGC_P, 10GGG_P and the 5TTT_P, 5GGG_P targets. In order to generate I-CreI derived coding sequence containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5′ end (amino acid positions 1-43) or the 3′ end (positions 39-167) of the I-CreI coding sequence. For both the 5′ and 3′ end, PCR amplification is carried out using primers specific to the vector (pCLS0542, FIG. 11 ): Gall OF or Ga110R and primers specific to the I-CreI coding sequence for amino acids 39-43 (assF 5′-ctannnttgaccttt-3′ (SEQ ID NO: 43) or assR 5′-aaaggtcaannntag-3′ (SEQ ID NO: 44)) where nnn codes for residue 40. The resulting PCR products contain 1 Sbp of homology with each other and approximately 100-200 bp of homology with the 2 micron-based replicative vectors, pCLS0542, marked with the LEU2 gene and pCLS1107, containing a kanamycin resistant gene.
Thus, to generate an intact coding sequence by in vivo homologous recombination in yeast, approximately 25 ng of each of the two overlapping PCR fragments and 25 ng of the pCLS0542 vector DNA linearized by digestion with NcoI and EagI or 25 ng of the pCLS1107 vector DNA linearized by digestion with DraIII and NgoMIV were used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200) using a high efficiency LiAc transformation protocol (Gietz, R. D. and R. A. Woods, Methods Enzymol, 2002, 350, 87-96). Combinatorial mutants can advantageously be generated as libraries: PCR reactions were pooled in equimolar amounts and transformed into yeast together with the linearized plasmid. Transformants were selected on either synthetic medium lacking leucine (pCLS0542) or rich medium containing G418 (PCLS1107). Colonies were picked using a colony picker (QpixII, Genetix), and grown in 96 well microtiter plates.
c) Mating of Meganuclease Expressing Clones and Screening in Yeast
The experimental procedure is as described in example 1, except that a low gridding density (about 4 spots/cm 2 ) was used.
c) Sequencing of Mutants
To recover the mutant expressing plasmids, yeast DNA was extracted using standard protocols and used to transform E. coli . Sequencing of mutant ORF was then performed on the plasmids by MILLEGEN SA. Alternatively, the ORFs of positive clones identified during the primary screening in yeast were amplified by PCR on yeast DNA extract from colonies (Akada et al., Biotechniques, 2000, 28(4): 668-70, 672, 674) using primers 5′ ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc 3′ (SEQ ID NO: 225) and 5′ggggaccactttgtacaagaaagctgggtttagtcggccgccgggaggatttcttcttctcgc 3′ (SEQ ID NO: 226) from PROLIGO and sequencing was performed directly on PCR product by MILLEGEN. PCR products were cloned either in (i) yeast gateway expression vectors harboring a galactose inducible promoter, LEU2 or KanR as selectable marker and a 2 micron origin of replication, (ii) CHO gateway expression vector pcDNA6.2 from INVITROGEN. Resulting clones were verified by sequencing (MILLEGEN).
B) Results
I-CreI N75 mutants cutting the 10TGC_P (ctgcacgtcgt_P) target and I-CreI N75 (Q44, R68, R70) mutants cutting the 5TTT_P (caaaactttgt_P) target were combined, resulting in combinatorial mutants that were screened against the Xa.3 target (ctgccttttgt_P).
I-CreI N75 mutants cutting the 10GGG_P target and I-CreI N75 mutants cutting the 5GGG_P target were combined, resulting in combinatorial mutants that were screened against the Xb.3 target.
At least twice the diversity of each library was screened. No correlation was observed between the number of active mutants identified and the number of combinations tested.
The Xc.3 target sequence contained the wild type sequence GTC at positions ±5, ±4 and ±3; therefore the combinatorial approach was not necessary to generate specific variants towards this target. I-CreI variants previously identified with altered substrate specificity towards bases ±10, ±9 and ±8 were directly screened against the Xc.3 DNA target.





TABLE XVI



Mutants used for combinatorial construction for the palindromic target Xx.3








Number of I-CreI


Number


mutants used for the


of combined


combinatorial reaction

Tested clones
unique

Targets
10NNN_P X 5NNN_P
diversity
(X diversity)
positive clones











Xa.3
510
na*
1099
2
8
(0.7%)


31
19

Xb.3
45
32
1440
2
30
(2%)

Xc.3
811
na*
811
na*
21
(2%)



na: non applicable

*only I-CreI variants with altered specificity towards nucleotides ±10, ±9 and ±8 were screened.






























Eight combinatorial mutants were found cleave the Xa.3 target (Table XVI). Two of the mutants cleaving the Xa.3 target have the following sequence:
I-CreI K28, N30, S33, R38, S40, S70 and N75 (called KNSRSSN), obtained by combination of I-CreI K28, N30, S33, R38, S40 and I-CreI S70,N75, and I-CreI A28, N30, S33, R38, K40, S70 and N75 (called ANSRKSN), obtained by combination of I-CreI K28, N30, S33, R38, K40 and I-CreI S70, N75.

Thirty combinatorial mutants were found to cleave the Xb.3 target (Table XVI).
Among the mutants cutting the 10GAG_P target which were tested, twenty one were found to cleave the Xc.3 target (Table XVI).
Results were confirmed in a secondary screen ( FIG. 12 ) and the predicted amino acid primary structure was confirmed by sequencing.
EXAMPLE 4
Making of Meganucleases Cleaving Xx.4
This example shows that I-CreI variants can cleave the Xx.4 DNA target sequence derived from the right part of the Xx.2 target in a palindromic form ( FIGS. 9 and 23 ). All target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleo-tides, followed by the suffix_P; for example, Xa.4 will be called taggatcctgt_P (SEQ ID NO: 37).
Xa.4 is similar to 5CCT_P in positions ±1, ±2, ±3, ±4, ±5 and ±7 and to 10AGG_P in positions ±1, ±2, ±7, ±8, ±9 and ±10. It was hypothesized that positions 16 and +11 would have little effect on the binding and cleavage activity.
Xb.4 is similar to 5TAC_P in positions ±1, ±2, ±3, ±4, ±5, ±6 and ±11 and to 10TGT_P in positions ±1, ±2, ±6, ±8, ±9, ±10, and ±11. It was hypothesized that positions ±7 would have little effect on the binding and cleavage activity.
Xc.4 is similar to 5TCT_P in positions ±1, ±2, ±3, ±4, ±5, ±7 and ±11 and to 10GTA_P in positions ±1, ±2, ±7, ±8, ±9, ±10, and ±11. It was hypothesized that positions +6 would have little effect on the binding and cleavage activity.
Mutants able to cleave the 5CCT_P, 5TAC_P and 5TCT_P targets were previously obtained by mutagenesis on I-CreI N75 at positions 44, 68 and 70 or I-CreI S70 at positions 44, 68, 75 and 77, as described in Arnould et al., J. Mol. Biol., 2006, 355, 443-458 ( FIG. 7 ). Mutants able to cleave the 10AGG_P, 10TGT_P, and 10GTA_P targets were obtained by mutagenesis on I-CreI N75 at positions 28, 30, 38, or 30, 33, 38, on I-CreI S70 N75 at positions 28, 33, 38 and 40 and 70, or on I-CreI D75 or N75 at two positions chosen from 28, 30, 32, 33, 38 and 40 (example 1 and FIG. 6 ). Thus, combining such pairs of mutants would allow for the cleavage of the Xx.4 target.
Some sets of proteins are both mutated at position 70. However, it was hypothesized that I-CreI comprises two separable functional subdomains. That implies that this position has little impact on the specificity in base 10 to 8 of the target.
Therefore, to check whether combined mutants could cleave the Xx.4 target, mutations at positions 44, 68, 70 and/or 75 from proteins cleaving 5CCT_P, 5TAC_P and 5TCT_P targets were combined with the 28, 30, 32, 33, 38, and/or 40 mutations from proteins cleaving 10AGG_P, 10TGT_P, and 10GTA_P targets, as illustrated in FIG. 24A .
A) Material and Methods
See example 3.
B) Results
I-CreI combined mutants were constructed by associating mutations at positions 44, 68 and 70 with the 28, 30, 33, 38 and 40 mutations on the I-CreI N75 scaffold. Combined mutants were screened against the Xx.4 DNA targets At least twice the diversity of each library was screened. No correlation was observed between the number of active mutants identified and the number of combinations tested. Two percent of the hybrid mutants appear to be functional for the Xb.4 DNA targets while as many as 55% were active against Xa.4. After secondary screening and sequencing, 104, 8 and 4 different cleavers were identified, for the Xa.4, Xb.4 and Xc.4 target, respectively (Table XVII).





TABLE XVII



Mutants used for combinatorial construction for the palindromic target Xx.4








Number of I-CreI


Number


mutants used for the


of combined


combinatorial reaction

Tested clones
unique

Targets
10NNN_P X 5NNN_P
diversity
(X diversity)
positive clones











Xa.4
9
21
189
4
104
(55%)

Xb.4
7
46
322
5
8
(2%)

Xc.4
3
47
141
3
24
(17%)



na: non applicable

*only I-CreI variants with altered specificity towards nucleotides 10, 9 and 8 were screened.





























I-CreI N75 mutants cutting the 10AGG_P (caggacgtcgt_P; SEQ ID NO: 32) target (amino acids at positions 28, 30, 33, 38 and 40 are indicated) and I-CreI N75 mutants cutting the 5CCT_P (caaaaccctgt_P; SEQ ID NO: 34) target (amino acids at positions 44, 68 and 70 are indicated) are listed in Table XVIII. 39 of the 104 positives are presented in FIG. 13 and Table XVIII.





TABLE XVIII



Cleavage of the Xa.4 target by the combined variants*





Amino acids at positions 28, 30, 33, 38 and 40 (ex: ENYRKstands for E28, N30, Y33, R38 and K40)














ENYRK
ENRRK
SNYRK
KGYGS
KGYHS
KGYRS
KGYTS
KDAHS
KDHKS
KDRGS

















Amino
KGA











acids at
KSN

positions
KQD





+

44, 68 and
KRE

+

+
+
+

70 (ex:
KNH

KGA stands
KSA

for K44,
KRN




+

G68 and
KGS




+
+

A70)
KNN




+


KNG


KGG





+


KTH





+


RRD


KRS



+
+


KRT


KSD




+
+


KSS
+

+


+


KHS




+
+


KTS

+


+
+


KRD

+
+

+
+


KAG




+
+


KAS

+
+

+


KAQ





+


KAN





+


KQS





+


KPS




+


KNA




+


KHD




+
+



+functional combination.

*all proteins have also a D75N mutation























































EXAMPLE 5
Making of Meganucleases Cleaving Xx.2 and Xx.1.1
I-CreI mutants able to cleave each of the palindromic Xx.2 derived targets Xx.3 and Xx.4, were identified in examples 3 and 4. A subset of pairs of such mutants (one cutting Xx.3 and one cutting Xx.4), were co-expressed in yeast. Upon co-expression, there should be three active molecular species, two homodimers, and one heterodimer. It was assayed whether the heterodimers that should be formed could cleave the Xx.1 and Xx.2 targets as depicted in FIG. 24A .
A) Material and Methods
a) Cloning of Mutants in Kanamycin Resistant Vector:
In order to co-express two I-CreI mutants in yeast, mutants cutting the Xx.3 sequence were subcloned in a kanamycin resistant yeast expression vector (PCLS1107, FIG. 14 ).
Mutants were amplified by PCR reaction using primers common for leucine vector (pCLS0542) and kanamycin vector (pCLS1107) (Ga110F and Ga110R). Approximately 25 ng of PCR fragment and 25 ng of vector DNA (pCLS1107) linearized by digestion with DraIII and NgoMIV are used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200) using a high efficiency LiAc transformation protocol. An intact coding sequence for the I-CreI mutant is generated by in vivo homologous recombination in yeast.
b) Mutants Coexpression:
Yeast strain expressing a mutant cutting the Xx.4 target was transformed with DNA coding for a mutant cutting the Xx.3 target in pCLS1107 expression vector. Transformants were selected on −L Glu+G418 medium.
c) Mating of Meganucleases Coexpressing Clones and Screening in Yeast:
The experimental procedure is as described in example 1, except that a low gridding density (about 4 spots/cm 2 ) was used.
B) Results:
The Table XIX summarizes the number of total and active heterodimers tested by co-expression in yeast against the targets Xx.2 and Xx.1.





TABLE XIX



Mutants used for the heterodimeric assay for each XPC target






Number of XPC



mutants used for
Number of positives


heterodimeric assay I
heterodimer combinations






Targets
target.3 × target.4
target.2
target.1









Xa
2
104
156
15

Xb
12
8
43
0

Xc
7
8
56
41
































In all cases, heterodimers with cleavage activity for the target.2 were identified ( FIGS. 15 a , 15 b and 24 B). All of the heterodimers tested were active against Xc.2 while 75% and 45% were able to cleave the Xa.2 and Xb.2 targets respectively. On the other hand, while the 4 central nucleotides sequences TTGA, GAAA, and ACAC found respectively in Xa.1, Xb.1 and Xc.1 targets did not impact the I-CreI cleavage activity when inserted in the I-CreI C1221 palindromic target, only a sub-fraction of the active heterodimers against Xa.2 and Xc.2 were able to cleave the Xa.1 and Xc.1 targets, respectively, while no cutters were found for the Xb.1 target ( FIGS. 15 a , 15 b and 24 B). The influence of the central sequence has been explained by its particular topology. Structure analysis revealed that this DNA region of the target is a region of maximal DNA bending with a curvature of around 50° resulting in base twisting and unstacking near the scissile phosphate groups (Chevalier et al., J. Mol. Biol., 2003329, 253-269). However, a precise mechanism explaining why certain sequences at the four central positions are compatible with cleavage activity and some others are not has still to be described.
Examples of functional combinations for the Xa.1 target are presented in Tables XX and XXI. As a general rule, functional heterodimers cutting Xx.1 sequence were always obtained when the two expressed proteins gave a strong signal as homodimer. Moreover, while many mutants are still very active against Xc.1, the mutants capable of cleaving the Xa.1 target displayed a weak activity





TABLE XX



Combinations that resulted in cleavage of the Xa.2 target (+) or Xa.2 and Xa.1(++) targets when expressed with KNSRSS





Amino acids at positions 28, 30, 33, 38 and 40 (ex: ENYRKstands for E28, N30, Y33, R38 and K40)














ENYRK
ENRRK
SNYRK
KGYGS
KGYHS
KGYRS
KGYTS
KDAHS
KDHKS
KDRGS

















Amino
KGA











acids at
KSN

positions
KQD





+

44, 68 and
KRE

+

+
++
++

70 (ex:
KNH

KGA stands
KSA

for K44,
KRN




+

G68 and
KGS




+
+

A70)
KNN




+


KNG


KGG





+


KTH





++


RRD


KRS



+
+


KRT


KSD




+
+


KSS
++

++


++


KHS




+


KTS




+
++


KRD

+
++

+
+


KAG




++


KAS

+
++

+


KAQ





+


KAN





+


KQS





+


KPS




+


KNA




+


KHD




+
+




























































TABLE XXI



Combinations that resulted in cleavage of the Xa.2 target (+) or Xa.2 and Xa.1 (++) targets when expressed with ANSRKS





Amino acids at positions 28, 30, 33, 38 and 40 (ex: ENYRKstands for E28, N30, Y33, R38 and K40)














ENYRK
ENRRK
SNYRK
KGYGS
KGYHS
KGYRS
KGYTS
KDAHS
KDHKS
KDRGS

















Amino
KGA











acids at
KSN

positions
KQD





+

44, 68 and
KRE

+

+
++
++

70 (ex:
KNH

KGA stands
KSA

for K44,
KRN

G68 and
KGS




+
+

A70)
KNN




+


KNG


KGG


KTH





++


RRD


KRS




+


KRT


KSD




+
+


KSS
++

++


++


KHS




+
+


KTS

+


+
++


KRD

+
++

+
+


KAG




++
+


KAS

+
++

+


KAQ





+


KAN





+


KQS





+


KPS




+


KNA




+


KHD




+
+























































EXAMPLE 6
Optimization of the Cleavage Activity of the Meganucleases
A) Material and Methods
Activity Improvement
Error-prone PCR was used to introduce random mutations in a pool of 4 chosen mutants. Libraries were generated by PCR using either Mn 2+ , or by two-steps process using dNTPs derivatives 8-oxo-dGTP and dPTP as described in the protocol from JENA BIOSCIENCE GmbH for the JBS dNTP-Mutagenesis kit. Primers used are: preATGCreFor (5′-gcataaattactatacttctatagacacgcaaacacaaatacacagcggccttgccacc-3′, SEQ ID NO: 227) and ICreIpostRev (5′-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgc-3′, SEQ ID NO: 228). For the first round of activity improvement, the new libraries were cloned in vivo in yeast in the linearized kanamycin vector (pCLS1107, FIG. 14 ) harboring a galactose inducible promoter, a KanR as selectable marker and a 2 micron origin of replication. For the second round of activity improvement, a 2 micron-based replicative vector marked with the LEU2 gene is used. Positives resulting clones were verified by sequencing (MILLEGEN).
B) Results
Since, the decrease in the cleavage activity between the target.2 and the target .1 ( FIG. 24B ) could be a consequence of a bias towards the 4 central nucleotides gtac introduced during the process of making meganucleases, it was hypothesized that a weak, activity could be subsequently improved by compensatory mutations. The FIG. 25 depicts the general workflow of the strategy, which was used to refine the proteins activity. The process of optimization of the cleavage activity is performed in two steps. First, after identification of the functional heterodimers for the target sequence Xa.1, a pool of 2 to 4 monomers initially identified by screening against Xa.4 target (mutant .4) is mutagenized by error-prone PCR, while its counterpart (e.g. the mutants identified by screening against the Xa.3 target) remain unmodified. The mutagenized library is then transformed in yeast and the screening is performed by mating with a yeast strain containing the target Xa.1 and the non-mutagenized mutant (active on Xa.3 in this case). About 2300 clones are usually tested, since this number of clones was sufficient to find mutants with improved activity. Once the combinations giving enhanced activity are identified, the same procedure is repeated to optimize the mutants interacting with the Xa.3 target (mutant .3). After error-prone PCR, the library is screened against Xa.1 target by mating with yeast containing the target and the improved mutants previously identified. At the end of this procedure, protein heterodimers are obtained, wherein both monomers have been optimized for a defined protein combination versus one target sequence. Finally, the improved variants are validated in a crossed experiment as shown in FIG. 26A (example of the cleavage activity in yeast of a subset of heterodimers against the target Xa.1 obtained after monomers improvements). Five improved mutants cleaving Xa.4 and 6 improved mutants cleaving Xa.3 were tested in combination with 2 and 8 improved mutants cleaving Xa.3 and Xa.4 respectively, against the Xa.1 target. The original mutants used for the error-prone PCR were incorporated in the experiment as controls. No activity could be detected against the target Xa.1 with homodimers alone as shown by the white colonies obtained after mating of yeast clones containing mutants cleaving only Xa.3 or Xa.4. In contrast, a strong cleavage activity can be visualized when mating occurred between yeast clones containing both mutant species indicating that the cleavage activity resulted from heterodimer formation. Furthermore, the strongest activity is achieved when both monomers have been optimized as compared to the activity achieved with heterodimers for which only one monomer activity has been improved.
The 6 combinations giving the strongest cleavage activity were selected and the ORFs were sequenced. Interestingly, the 6 most active heterodimers resulted from 6 different combinations of 3 independent mutants cleaving Xa.3 with 2 different mutants cleaving Xa.4. The FIG. 26B shows the sequence of the original proteins used for the improvement process (I1-I4)) and their respective optimized proteins (M1-M5). The 15 and 16 sequences in FIG. 26B are the protein sequences of the 2 monomers giving the best activity on Xc.1 target by co-expression assay. As the cleavage of this target was highly efficient, no further activity refinement was needed.
The protein sequence analysis of the best cutters does not reveal any particular protein domains affected by the mutation process. As compared with the original sequence, the error-prone PCR introduced mutations at positions 19, 69 and 87 in the ORF of active mutants cleaving Xa.3 target and mutations at positions 32, 85 and 109 in the coding sequence of the mutants cleaving Xa.4 target. The positions 33 and 38 were also reverted in the protein sequence of the M2 and M5 proteins. Interestingly, the amino acid at position 19 was mutated in all proteins with activity towards the Xa.3 target. This position is part of the catalytic site (Chevalier et al., Biochemistry, 2004, 43, 14015-14026) and with the adjacent Asp20, is involved in the metal cation binding. This is the only mutation which can be directly linked to improvement of the catalytic mechanism. The mutations in positions 32 and 69 affect the protein-DNA interface and the mutations in positions 85 and 87 affect the hydrophobic core. The other mutations affect mainly the core protein indicating that a mechanism of propagated conformational change is responsible for the improved activity. This long range effect could, for example, improve the binding affinity of the mutant and therefore increase its cleavage activity.
These results demonstrate that the combinatorial approach associated to random mutagenesis allows the rapid and efficient production of custom-designed endonucleases for specific DNA substrates.
EXAMPLE 7
The Heterodimeric Meganucleases are Functional in Mammalian Cells
A) Material and Methods
Mammalian Cells Assays
CHO cells were transfected with Polyfect® transfection reagent according to the supplier (QIAGEN) protocol. 72 hours after transfection, culture medium was removed and 150 μl of lysis/revelation buffer added for β-galactosidase liquid assay (1 liter of buffer contains 100 ml of lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton X100 0.1%, BSA 0.1 mg/ml, protease inhibitors), 10 ml of Mg 100× buffer (MgCl 2 100 mM, β-mercaptoethanol 35%), 110 ml ONPG (8 mg/ml) and 780 ml of sodium phosphate 0.1 M pH7.5). After incubation at 37° C., OD was measured at 42 0 nm. The entire process is performed on an automated Velocity11 BioCel platform.
B) Results
The hybrid meganucleases active in yeast were tested in mammalian cells by transient co-transfection of CHO cells with a target vector and meganuclease expression vectors. For this purpose, subsets of mutants and their corresponding targets (Xa.1 and Xc.1) were cloned into appropriate vectors as described in example 1, and the meganuclease-induced recombination efficiency was measured by a standard, quantitative ONPG assay that monitors the restoration of a functional β-galactosidase gene, as described previously (International PCT Application WO 2006/097853; Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Smith et al., Nucleic Acids Res., Epub 27 November 2006). FIG. 27 shows the cleavage activity in CHO-K1 cells for the most active hybrid proteins identified in yeast. All target plasmids carry a minimal I-SceI site of 18 bp as an internal control; therefore all mutants can be compared to the I-SceI activity in the same experimental conditions. This 18 bp I-SceI site is weakly cleaved by the I-SceI protein and was chosen in order to avoid saturation of the signal in CHO cells. No cleavage activity could be detected when a single protein is expressed as judged by their background level activity ( FIG. 27 ; Ø/Ø, Ø/I1 to Ø/M3, and Ø/I3 to Ø/M5) indicating that activity is dependent on heterodimer formation. Furthermore, the co-expression of the initial combinatorial mutants (I1, I3) has a very weak activity which is virtually not distinguishable from the background level. The cleavage activity increases as soon as one of the protein partners has been improved. Finally, the most efficient cleavage activities for the Xa.1 target is achieved by co-expression of 2 improved proteins and reaches an activity level similar to the activity of the mutants identified for the Xc. 1 target. These results confirm the data obtained in yeast in which co-expression of the initial combinatorial mutants lead to efficient cleavage of the Xc.1 target without the need of improvement activity while the mutants directed towards Xa.1 had poor activity. However a high activity for the Xa.1 target could be generated after introduction of compensatory mutations. When monomers are expressed on their own, neither toxicity nor cleavage, as shown in FIG. 27 , could be detected, showing that the mutants designed towards Xa and Xc keep the original specificity of the I-CreI homing endonuclease or at least a specificity compatible with cell survival.
EXAMPLE 8
Analysis of the Individually Mutated Target Cleavage Pattern
The degeneracy at individual positions of the I-CreI target has been previously assayed using in vitro site selection in which variant DNA targets cleavable by wtI-CreI could be recovered (Argast et al., J. Mol. Biol., 1998, 280, 345-353). It indicates that most nucleotide positions in the site can be mutated without loss of binding or cleavage. However no exhaustive study was done. In order to compare the improved mutants towards Xa.1 target with I-CreI homing endonuclease, we have generated all possible targets carrying individual mutation were generated for Xa.3, Xa.4 and the palindromic I-CreI target C1221. The protein scaffold used to generate all the mutants carries a D to N mutation at position 75. This mutation was introduced in order to decrease the energetic strains caused by the replacement of the basic residues at positions 68 and 70 in the libraries. It was shown previously that the D75N mutation decreased the toxicity of over-expressed I-CreI protein without affecting the protein basic folding properties and activity (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). The extent of degeneracy of base-pair recognition of wild type I-CreI (wt I-CreI), the initial protein scaffold I-CreI(N75) and the combinatorial mutants was assayed by measuring their cleavage efficiency in yeast on their respective palindromic targets (C1221, Xa.3 and Xa.4) carrying individual site mutations. I-CreI D75 (wt) and I-CreI N75
As shown in FIG. 28A , the wild type protein (I-CreI-D75) can accommodate many individual mutations on its palindromic target (the font size of each nucleotide is proportional to the cleavage efficiency). The positions ±11, ±8, ±2, and ±1 are the most tolerant positions since none of the 4 nucleotides at these positions affect the cleavage efficiency. In contrast, positions ±9 and ±3 accept very few changes. In comparison, the I-CreI scaffold used to generate the mutants (I-CreI-N75) reveals a different pattern and seems to be less tolerant to point mutations in its target. The most stringent differences are seen at positions ±1, ±3, ±10, and ±1. The only bases allowing target cleavage are ±1T, ±9A and ±10A while G and A at position ±11 inhibit the cleavage of the target by I-CreI(N75) protein. For both wt I-CreI and I-CreI(N75), C or A and T or C at positions ±7 and ±6 respectively, allow maximum cleavage efficiency. Altogether wt I-CreI and I-CreI(N75) cleave respectively 26 and 14 targets carrying single mutation. Based on this data, and if it is assumed that each half target can have an independent impact on the global cleavage efficiency, wt I-CreI should be able to cleave a maximum of 676 (26×26) out of 1156 (34×34) non palindromic targets with individual mutations while I-CreN75 should cut only 196 (14×14). Probably, the restricted pattern of the I-Cre(N75) observed in this study could explain the absence of toxicity of this I-CreI variant. As suggested in early studies (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269; Seligman et al., Genetics, 1997, 147, 1353-1664), the tolerance of individual nucleotide polymorphisms allows I-CreI(D75) to recognize a defined population of targets and facilitates the repeated horizontal transmission of the intron during evolution.
Combinatorial Mutants Cleaving the Xa.3 and Xa.4 Targets
The FIG. 28B shows the results obtained with the best combinatorial mutants cleaving the Xa.3 and Xa.4 targets. The mutant cleaving the Xa.4 target is much more permissive than the mutant cleaving the Xa.3 target for individual mutation on their respective targets. The combinatorial Xa.4 mutant is able to cleave 24 palindromic targets of which 20 are strongly cleaved. The pattern of single-base substitutions tolerated by the Xa.4 mutants is very similar to that of wt I-CreI towards its own target. In contrast, custom-designed variant towards Xa.3 targets revealed to have the highest stringency regarding individual mutations as only 3 palindromic targets are highly cleavable. This mutant is capable of cleaving only 8 (of which 4 are efficiently cleaved) out of the 34 palindromic targets. Except at the position ±5 which can tolerate G and T, efficient cleavage is achieved only for the target on which it has been selected. As none of the mutants tested displayed degeneracy greater than the wt I-CreI natural protein, these data provide evidence that the combinatorial approach used to generate the mutants results in a change of substrate specificity instead of a simple relaxation of the protein specificity towards its target. Furthermore, the combinatorial mutants have been checked against the parental sequences and the original I-CreI target sequence and none of the proteins tested cleaves these targets. Altogether, the heterodimer designed to cleave the Xa.1 target should be able to cut a maximum of 192 (8×24) out of the 1156 non palindromic targets with individual mutations.