WO2012138901A1 - Procédé pour l'amélioration de l'efficacité d'endonucléases à coupure rare et ses utilisations - Google Patents

Procédé pour l'amélioration de l'efficacité d'endonucléases à coupure rare et ses utilisations Download PDF

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WO2012138901A1
WO2012138901A1 PCT/US2012/032386 US2012032386W WO2012138901A1 WO 2012138901 A1 WO2012138901 A1 WO 2012138901A1 US 2012032386 W US2012032386 W US 2012032386W WO 2012138901 A1 WO2012138901 A1 WO 2012138901A1
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rare
cutting endonuclease
cutting
endonuclease
chimeric
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George H. Silva
Claudia BERTONATI
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Cellectis SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a method for enhancing the efficiency of rare-cutting endonucleases such as meganucleases. More specifically, the present invention concerns a method for the creation of fusion proteins that consist of one or more "enhancer" domains fused to at least one rare-cutting endonuclease-derived scaffold in a single polypeptide chain for simple and efficient vectorization.
  • the present invention also relates to engineered chimeric rare-cutting endonucleases, vectors, compositions and kits used to implement the method and use of said chimeric rare-cutting endonucleases according to the invention for various applications ranging from homologous gene targeting to targeted mutagenesis and sequence removal.
  • DSBs double-strand breaks
  • HR homologous recombination
  • SSA Single-strand annealing
  • NHEJ non-homologous end joining
  • HCT Homologous gene targeting
  • Orr- Weaver Szostak et al. 1981
  • Orr-Weaver Szostak et al. 1983
  • Rothstein 1983 was one of the first methods for rational genome engineering and remains to this day a standard for the generation of engineered cells or knock-out mice (Capecchi 2001).
  • An inherently low efficiency has nevertheless prevented it from being used as a routine protocol in most cell types and organisms.
  • DSBs induced by biological reagents e.g. meganucleases, ZFNs and TALENs (see below), which cleave DNA by hydrolysis of two phosphodiester bonds
  • the DNA can be rejoined in a seamless manner by simple re-ligationof the cohesive ends.
  • deleterious insertions or deletions indels of various sizes can occur at the breaks, eventually resulting in gene inactivation (Liang, Han et al. 1998; Lloyd, Plaisier et al. 2005; Doyon, McCammon et al. 2008; Perez, Wang et al. 2008; Santiago, Chan et al. 2008; Kim, Lee et al.
  • Targeted mutagenesis based on NEHJ has been used to trigger inactivation of single or even multiple genes in immortalized cell lines (Cost, Freyvert et al. 2010; Liu, Chan et al. 2010).
  • this method opens new perspectives for organisms in which the classical HR-based gene knock-out methods have proven inefficient, or at least difficult to establish (Doyon, McCammon et al. 2008; Geurts, Cost et al. 2009; Shukla, Doyon et al. 2009; Yang, Djukanovic et al. 2009; Gao, Smith et al. 2010; Mashimo, Takizawa et al. 2010; Menoret, Iscache et al. 2010).
  • Meganucleases also called homing endonucleases (HEs) can be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and PD-(D/E)XK (Stoddard 2005; Zhao, Bonocora et al. 2007). Structural data are available for at least one member of each family. The most well studied family is that of the LAGLIDADG proteins, with a considerable body of biochemical, genetic and structural work having established that these endonucleases could be used as molecular tools (Stoddard, Scharenberg et al. 2007; Arnould, Delenda et al. 2011).
  • Member proteins are composed of domains that adopt a similar ⁇ fold, with the LAGLIDADG motif comprising the terminal region of the first helix and not only contributing to a bipartite catalytic center but also forming the core subunit/subunit interaction (Stoddard 2005). Two such ⁇ / ⁇ domains assemble to form the functional protein, with the ⁇ -strands in each creating a saddle- shaped DNA binding region. The spatial separation of the catalytic center with regions directly interacting with the DNA has allowed for specificity re-engineering (Seligman, Chisholm et al. 2002; Sussman, Chadsey et al. 2004; Arnould, Chames et al.
  • LAGLIDADG HEs members from two other families, GIY-YIG and HNH, are of particular interest. Biochemical and structural studies have established that in both families, member proteins can adopt a bipartite fold with distinct functional domains: (1) a catalytic domain responsible mainly for DNA cleavage, and; (2) a DNA-binding domain to provide target specificity (Stoddard 2005; Marcaida, Munoz et al. 2010).
  • the related GIY-YIG HEs I- TevI and l-Bmol have been exploited to demonstrate the interchangeability of the DNA-binding region for these enzymes (Liu, Derbyshire et al. 2006).
  • IENR1 intron-encoded endonuclease repeat motif found in endonucleases of the GIY-YIG family.
  • the catalytic head of l-Basl has sequence similarity to those of the HNH HEs l-Hmul, l-Hmull and l-Twol, all of which function as strand-specific nickases (Landthaler, Begley et al. 2002; Landthaler and Shub 2003; Landthaler, Lau et al.
  • HNH motif has also been identified in nonspecific nucleases such the E.coli colicins (e.g. ColE9 and ColE7), EndA from S. pneumoniae, NucA from Anabaena and CAD (Midon, Schafer et al. 2011).
  • E.coli colicins e.g. ColE9 and ColE7
  • EndA from S. pneumoniae
  • NucA from Anabaena
  • CAD Mordon, Schafer et al. 2011
  • several of these nucleases contain the signature DRGH motif and share structural homology with core elements forming the ⁇ -Me-finger active site motif.
  • Zinc-finger nucleases generated by fusing Zinc-finger-based DNA-binding domains to an independent catalytic domain via a flexible linker (Kim, Cha et al. 1996; Smith, Berg et al. 1999; Smith, Bibikova et al. 2000), represent another type of engineered nuclease commonly used to stimulate gene targeting.
  • the archetypal ZFNs are based on the catalytic domain of the Type IIS restriction enzyme Fokl and have been successfully used to induce gene correction, gene insertion, and gene deletion.
  • Zinc Finger-based DNA binding domains are made of strings of 3 or 4 individual Zinc Fingers, each recognizing a DNA triplet (Pabo, Peisach et al. 2001).
  • ZFNs are easy to design, using combinatorial assembly of preexisting Zinc Fingers with known recognition patterns (Choo and Klug 1994; Choo and Klug 1994; Kim, Lee et al. 2009).
  • close examination of high resolution structures shows that there are actually crosstalks between units (EIrod-Erickson, Rould et al. 1996), and several methods have been used to assemble ZF proteins by choosing individual Zinc Fingers in a context dependant manner (Greisman and Pabo 1997; Isalan and Choo 2001; Maeder, Thibodeau-Beganny et al. 2008; Ramirez, Foley et al. 2008) to achieve better success rates and reagents of better quality.
  • TALE Transcription Activator Like Effectors
  • each base pair in the DNA target is contacted by a single repeat, with the specificity resulting from the two variant amino acids of the repeat (the so-called repeat variable dipeptide, RVD).
  • RVD repeat variable dipeptide
  • TALEN Fokl-based TALE-nuclease
  • the functional layout of a Fokl-based TALE-nuclease is essentially that of a ZFN, with the Zinc-finger DNA binding domain being replaced by the TALE domain (Christian, Cermak et al. 2010; Li, Huang et al. 2011).
  • DNA cleavage by a TALEN requires two DNA recognition regions flanking an unspecific central region. This central "spacer" DNA region is essential to promote catalysis by the dimerizing Fokl catalytic domain, and extensive effort has been placed into optimizing the distance between the DNA binding sites (Christian, Cermak et al. 2010; Miller, Tan et al. 2011).
  • the length of the spacer has been varied from 14 to 30 base pairs, with efficacy in DNA cleavage being interdependent with spacer length as well as TALE scaffold construction (i.e. the nature of the fusion construct used). It is still unknown whether differences in the repeat region (i.e. RVD type and number used) have an impact on the DNA "spacer” requirements or on the efficacy of DNA cleavage by TALENs. Nevertheless, TALE-nucleases have been shown to be active to various extents in cell-based assays in yeast, mammalian cells and plants (Christian, Cermak et al. 2010; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011).
  • the efficiency of rare-cutting endonuclease e.g. their ability to induce a desired event (Homologous gene targeting, targeted mutagenesis, sequence removal or excision) at a locus, depends on several parameters, including the specific activity of the nuclease, probably the accessibility of the target, and the efficacy and outcome of the repair pathway(s) resulting in the desired event (homologous repair for gene targeting, NHEJ pathways for targeted mutagenesis).
  • Nucleases (Smith, Bibikova et al. 2000). These ends, which result from hydrolysis of phosphodiester bonds, can be re-ligated in vivo by NHEJ in a seamless way (i.e a scarless re-ligation).
  • the restoration of a cleavable target sequence allows for a new cleavage event by the same endonuclease, and thus, a series of futile cycles of cleavage and re-ligation events can take place.
  • Indirect evidences have shown that even in the yeast Saccharomyces cerevisiae, such cycles could take place upon continuous cleavage by the HO endonuclease (Lee, Paques et al. 1999).
  • DSB resection is important for certain DSB pathways. Extensive DSB resection, resulting in the generation of large single stranded regions (a few hundred nucleotides at least), has been shown in yeast to initiate single strand annealing (Sugawara and Haber 1992) and strand invasion, the ATP-dependant step that initiates many homologous recombination events of DNA duplex invasion by an homologous strand that (White and Haber 1990; Sun, Treco et al. 1991) (for a review of mechanisms, see (Paques and Haber 1999)).
  • DSB resection depends on several proteins including BLM/Sgsl and DNA2, EXOI, and the MRN complex (Mrell, Rad50, Nbsl/Xrs2) and is thought to result from different pathways. MRN is involved in a small scale resection process, while two redundant pathways depending on BLM and DNA2 on one hand, and on EXOI on another hand, would be involved in extensive resection (Mimitou and Symington 2008; Nimonkar, Genschel et al. 2011).
  • the authors of the present invention have developed a method to significantly enhance the efficiency of rare-cutting endonucleases such as LAGLIDADG-type meganucleases.
  • the use of novel "enhancer" domains allows for boosting the overall efficiency of the meganuclease without modifying its specificity determinants (i.e. residues making base-specific contacts to the DNA).
  • the invention allows for generating several distinct types of enzymes that can be applied to applications ranging from homologous gene targeting to targeted mutagenesis and sequence removal.
  • the present invention relates to a method for enhancing the efficiency of a rare- cutting endonuclease for a DNA target sequence by merging a rare-cutting endonuclease-derived scaffold with at least one enhancer domain to obtain a chimeric rare-cutting endonuclease with an enhanced efficiency for said DNA target sequence.
  • the present invention concerns a method for the creation of fusion proteins that consist of engineering a fusion protein between a rare-cutting endonuclease-derived scaffold and at least one enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease when fused to it.
  • the present invention also concerns the creation of functional single polypeptide fusion proteins for simple and efficient vectorization.
  • the present invention relates to chimeric rare- cutting endonucleases comprising at least an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease when fused to it, thereby obtaining a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence compared to a corresponding rare-cutting endonuclease lacking said enhancer domain.
  • the present invention also relates to engineered rare-cutting endonucleases, vectors, compositions and kits used to implement the method and use of said chimeric rare-cutting endonucleases according to the invention for various applications ranging from homologous gene targeting to targeted mutagenesis and sequence removal.
  • Figure 1 Endonuclease-induced gene targeting approaches. Upon cleavage, DNA repair mechanisms may result in one of several outcomes.
  • A When a double-strand break is targeted between two direct repeats, HR can result in the deletion of one repeat together with the intervening sequence.
  • Gene insertion (B) or correction (C) can be achieved by the introduction of a DNA repair matrix containing sequences homologous to the endogenous sequence surrounding the DNA break. Mutations can be corrected either at or distal to the break, with the frequency of correction decreasing with increasing distance.
  • D The misrepair of DNA ends by error-prone NHEJ can result in insertions or deletions of various sizes, leading to gene inactivation.
  • Figure 2 Sequences of target DNA recognized by l-Crel.
  • C1234 (SEQ ID NO: 3) represents the partially symmetric DNA sequence recognized and cleaved by wild-type l-Crel.
  • C1221 (SEQ ID NO: 2) represents a palindromic DNA sequence, derived from C1234 (SEQ ID NO: 3), recognized and cleaved by the l-Crel meganuclease. Nucleotides are numbered outward (-/+) from the center of the target. The nature of the nucleotides at positions -2 to +2 can potentially interfere with the cleavage activity of the protein.
  • FIG. 3 Schematic of enhanced rare-cutting endonucleases, such as meganucleases, fusion configurations.
  • Rare-cutting endonucleases such as meganucleases, can be enhanced through the addition of a domain to promote existing or alternate activities.
  • the order (N- v.s C-terminal) of addition and number of the enhancer domains can vary with the application.
  • Enhanced fusion construct are optimized to address or overcome distinct problems.
  • A The addition of two enhancer domains to an active meganuclease can enhance DNA binding and/or cleavage activity.
  • Such a configuration can be achieved via (i) a single N- or C-terminal fusion to a homodimeric variant; (ii) a single N- or C-terminal fusion to individual monomers of a heterodimer, or; (iii) a double fusion to a monomeric protein.
  • B When specificity reengineering precludes maintaining cleavage activity of the meganuclease, the attached enhancer domains can provide alternative functions.
  • C) and (D) represent instances of (A) and (B), respectively, when only one enhancer domain is needed or tolerated per fusion protein (e.g. either as an N- or C-terminal fusion or in the context of a single-chain molecule). Fusion junctions (N- vs. C- terminal) and linker designs can vary with the application. Components of the fusion proteins are listed in the legend.
  • FIG. 4 Schematic of DNA cleavage, in vivo re-ligation and other repair pathways.
  • cleavage by peptidic rare-cutting endonucleases usually result in a DNA double strand break (DSB) with cohesive ends.
  • DSB DNA double strand break
  • meganucleases from the LAGLIDADG family such as l-Scel and I- Crel, produce DSBs with 3' overhang.
  • These cohesive ends can be re-ligated in vivo by NHEJ, resulting in seamless repair, and in the restoration of a cleavable target sequence, which can in turn be processed again by the same endonuclease.
  • a series of futile cycles of cleavage and re-ligation events can take place.
  • Imprecise NHEJ or homologous recombination can alter or remove the cleavage site, resulting in cycle exit (A).
  • Two other ways can also stop the process : (i) Chromosome loss can occur as the consequence of failure to repair the DSB; (ii) a loss of nuclease (degradation, dilution, cell division, etc.).
  • B-E Consequences of cleavage of additional phosphodiester bonds. The addition of a single nickase activity (B) or of two nickase activities affecting the same strand (C) would result in a single strand gap, and suppress the cohesive ends, which could in turn affect the spectrum of events.
  • the present invention relates to a method for enhancing the efficiency of a rare- cutting endonuclease for a DNA target sequence by merging a rare-cutting endonuclease-derived scaffold with at least one enhancer domain to obtain a chimeric rare-cutting endonuclease with an enhanced efficiency for said DNA target sequence.
  • a first aspect of the present invention is a method for enhancing rare-cutting endonuclease efficiency at a genomic locus of interest in a cell comprising the steps of:
  • said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold. In another preferred embodiment, said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold. In another embodiment, two enhancer domains are fused to both N-terminus part of said rare-cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived monomer scaffold of a homodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising two or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived monomer scaffold of a heterodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two or three or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived scaffold from a single-chain endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two enhancer domains.
  • said enhancer domain is catalytically active, or not providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a protein domain derived from an exonuclease.
  • said enhancer domain consists of a protein domain derived from an endonuclease.
  • said enhancer domain consists of a protein domain derived from a cleavase.
  • said enhancer domain consists of a protein domain derived from a nickase.
  • said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), .HinPll, l-Basl, I- Bmol, l-Hmul, l-Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB, Endodeoxyribonuclease I (
  • said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112).
  • any combinations of two protein domains selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinPll, l-Basl, l-Bmol, l-Hmul, I- Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonu
  • l-Hmul catalytic domain can be fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold and ColE7 can be fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a catalytically active derivative of the protein domains listed above and in Table 1, providing functional and/or structural support to said rare-cutting endonuclease- derived scaffold.
  • said enhancer domain consists of a catalytically inactive derivative of the protein domains listed above and in Table 1, providing structural support to said rare-cutting endonuclease-derived scaffold.
  • said rare-cutting endonuclease-derived scaffold is derived from a meganuclease.
  • said meganuclease comprises two identical monomers.
  • said meganuclease comprises two non-identical monomers.
  • said meganuclease is a single-chain meganuclease.
  • said rare-cutting endonuclease-derived scaffold is derived from the group consisting of l-Crel, a functional mutant of l-Crel, a variant of l-Crel or a derivative thereof.
  • rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 or residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or two or three or four or five or six or seven or eight or nine or ten further amino acid mutations.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and K82A mutation.
  • said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of l-Crel_NFSl (SEQ ID NO: 6); l-Crel_NFS2 (SEQ ID NO: 7); and l-Crel_CFSl (SEQ ID NO: 8).
  • said chimeric rare-cutting endonuclease can comprise at least one peptidic linker between said rare-cutting endonuclease-derived scaffold and said at least one enhancer domain.
  • said peptidic linker sequence is selected from the group consisting of
  • the peptidic linker that can link said enhancer domain to the rare-cutting endonuclease-derived scaffold according to the method of the present invention can be selected from the group consisting of NFSl (SEQ ID NO: 98), NFS2 (SEQ ID NO: 99) and CFSl (SEQ ID NO: 100).
  • NFSl SEQ ID NO: 98
  • NFS2 SEQ ID NO: 99
  • CFSl SEQ ID NO: 100.
  • a peptidic linker is not needed to fuse said enhancer domain to said rare-cutting endonuclease-derived scaffold in order to obtain a chimeric rare-cutting endonuclease according to the present invention.
  • Table 2 List of peptidic linkers that can be used in chimeric rare-cutting endonuclease.
  • Enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, can also be a consequence of a fuctional support by said at least one enhancer domain.
  • said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a cleavase. In another more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nickase. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an exonuclease.
  • enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention can be the hydrolysis of additional phosphodiester bonds at the cleavage site to promote exit from such futile cycles of cleavage and re-ligation events via imprecise NHEJ or Homologous Recombination or Single Strand Annealing (SSA).
  • SSA Single Strand Annealing
  • Said hydrolysis of additional phosphodiester bonds at the cleavage site by said at least one enhancer according to the invention can lead to different types of DSB resection affecting at said DSB cleavage site, one single DNA strand or both DNA strands, affecting either 5' overhangs ends, either 3' overhangs ends, or both ends and depending on the length of said resection.
  • adding new nickase or cleavase activities to the existing cleavase activity of a rare-cutting endonuclease enhances the efficiency of the resulting chimeric rare-cutting endonuclease according to the invention, at a genomic locus of interest.
  • the addition of a single nickase activity can result in a single strand gap, and suppress the cohesivity of the ends, which can also enhances the efficiency of the resulting chimeric rare-cutting endonuclease at a genomic locus of interest, according to the invention, via stimulation of one or several alternative repair outcomes mentioned above.
  • the method according to the invention enhances rare-cutting endonuclease efficiency for a DNA target sequence.
  • Enhancement of efficiency of a rare-cutting endonuclease according to the present invention can be a consequence of a structural support by said at least one enhancer domain.
  • said structural support enhances the binding of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as a non-limiting example.
  • said structural support by at least one enhancer domain enhances the existing catalytical activity of a chimeric rare-cutting
  • said at least one enhancer domain enhances both the binding and the existing catalytical activity of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as another non-limiting example. All these non- limiting examples lead to a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target at a genomic locus of interest, according to the present invention.
  • the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention.
  • the addition of one nickase domain to an active rare- cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one nickase activity (depicted in figure 4B).
  • the addition of two nickase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two nickase activities (as depicted in figures 5C and 5D).
  • the addition of one cleavase domain to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one cleavase activity (figure 4E).
  • the addition of two cleavase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two cleavase activities.
  • enhancement of efficiency of a rare-cutting endonuclease refers to the increase in the detected level of said efficiency, against a target DNA sequence, of a second rare-cutting endonuclease in comparison to the activity of a first rare-cutting endonuclease against the same target DNA sequence.
  • Said second rare-cutting endonuclease can be an engineered rare-cutting endonuclease, i.e. a chimeric rare-cutting endonuclease according to the invention or not.
  • Said first rare-cutting endonuclease can be a wild-type rare-cutting endonuclease, a starting rare-cutting endonuclease, an engineered rare-cutting endonuclease i.e a chimeric rare-cutting
  • Said enhancement of efficiency can be determined, as non-limiting example, by measuring the level of cleavage-induced recombination generated by said rare-cutting endonuclease or said chimeric rare-cutting endonuclease according to the present invention. Said level of cleavage-induced recombination can be measured by a cell-based recombination assay as described in the International PCT Application WO 2004/067736, as non-limiting example.
  • enhancement of efficacy in cells can be, but is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays.
  • additional phosphodiesterase activities as described in Figure 4 could barely affect the cleavage profile, as detected by in vitro cleavage and separation of the cleavage products on an electrophoresis gel.
  • the DSB ends generated in this way could be more prone to induce detectable genomic rearrangements such as targeted mutagenesis (by imprecise NHEJ) or homologous recombination.
  • Said enhancement in efficacy of said rare-cutting endonuclease is at least a 5% enhancement compared to the starting scaffold, more preferably at least a 10% enhancement, again more preferably at least a 15% enhancement, again more preferably at least a 20% enhancement, again more preferably at least a 25% enhancement, again more preferably a 50% enhancement, again more preferably a enhancement greater than 50%.
  • the enhancement of efficiency of a rare-cutting endonuclease according to the method of the present invention can also be an enhancement in the efficiency for 2NN derivatives of a DNA target sequence by a rare-cutting endonuclease from the LAGLIDADG meganuclease subfamily.
  • 2NN derivatives of a DNA target sequence is intended variants of a given DNA target sequence having mutations in the four central nucleotides -2 to +2 as numbered in figure 2.
  • the method according to the invention enhances the efficiency for derivatives of the DNA target sequence C1221 (SEQ ID NO: 2) having mutations in the four central nucleotides -2 to +2 as numbered in figure 2, i.e that do not have GTAC sequence at position -2 to +2 as numbered in figure 2.
  • the method according to the invention allows the recombination to be initiated at 2NN derivatives that are not normally processed by a starting rare- cutting endonuclease scaffold from the LAGLIDADG meganuclease subfamily.
  • the method according to the invention allows the processing of 2NN derivatives that have - 2 to +2 target sequences as listed in Table 4 and Table 5, non-originally cleaved by l-Crel.
  • this first aspect of the invention is the method according to the invention wherein said chimeric rare-cutting endonuclease is selected from the group consisting of hHmuCre_D0101 (SEQ ID NO: 9), hHmuCre_D0201 (SEQ ID NO: 105), hHmuCre_D0301 (SEQ ID NO: 106), hHmuCre_D0401 (SEQ ID NO: 107), hHmuCre_D0102 (SEQ ID NO: 108), hHmuCre_D0202 (SEQ ID NO: 109), hHmuCre_D0302 (SEQ ID NO: 110) and hHmuCre_D0402 (SEQ ID NO: 111).
  • efficiency of rare-cutting endonucleases such as meganucleases can be enhanced through the addition of a domain to promote existing or alternate activities.
  • the order (N- v.s C- terminal) of addition and number of the enhancer domains can vary with the application as depicted in Figure 3.
  • Enhanced fusion construct are optimized to address or overcome distinct problems.
  • Figure 3A The addition of two enhancer domains to an active rare-cutting endonuclease such as a meganuclease can enhance DNA binding and/or cleavage activity.
  • Such a configuration can be achieved via (i) a single N- or C-terminal fusion to a homodimeric variant; (ii) a single N- or C-terminal fusion to individual monomers of a heterodimer, or; (iii) a double fusion to a monomeric protein.
  • Figure 3B When specificity reengineering precludes maintaining cleavage activity of the rare- cutting endonuclease such as a meganuclease, the attached enhancer domains can provide alternative functions.
  • Figure 3C) and (Figure 3D) represent instances of ( Figure 3A) and ( Figure 3B), respectively, when only one enhancer domain is needed or tolerated per fusion protein (e.g. either as an N- or C-terminal fusion or in the context of a single-chain molecule). Fusion junctions (N- vs. C- terminal) and linker designs can vary with the application.
  • the present invention can relate to a method for increasing targeted H (and NHEJ) when efficiency is enhanced in a chimeric rare-cutting endonuclease targeting a DNA target sequence according to the invention.
  • said efficiency can be enhanced by the addition of a catalytically active cleavase enhancer domain according to the invention, providing functional and/or structural support to the starting rare-cutting endonuclease scaffold according to the invention.
  • said efficiency can be enhanced by the addition of two catalytically active cleavase enhancer domains according to the invention, providing functional and/or structural support to the starting rare-cutting endonuclease scaffold according to the invention.
  • two cataiyticaily active cleavase enhancer domains can allow an increase in Double-strand break-induced mutagenesis by leading to a loss of genetic information between two nearby DNA double strand breaks and thus preventing any scarless re-ligation of targeted genomic locus of interest by NHEJ.
  • the present invention can relate to a method for increasing targeted Homologous Recombination (HR) with less NHEJ (i.e in a more conservative fashion) when at least one single-strand break activity is promoted in a chimeric rare-cutting endonuclease targeting a DNA target sequence according to the invention.
  • HR Homologous Recombination
  • NHEJ i.e in a more conservative fashion
  • the efficiency of said chimeric rare-cutting endonuclease could be enhanced by the addition of a cataiyticaily active nickase enhancer domain according to the invention.
  • the efficiency of said chimeric rare-cutting endonuclease could be enhanced by the addition of two cataiyticaily active nickase enhancer domains according to the invention, all nickase enhancer domains being able to cut the same DNA strand. More preferably, the addition of a least one nickase enhancer domain according to the invention can be performed on an inactive rare- cutting endonuclease scaffold which serves as a scaffold for targeted DNA binding.
  • the present invention can relate to a method for increasing excision of a single-strand of DNA spanning the starting rare-cutting endonuclease DNA binding region when both one cleavase enhancer domain and one nickase enhancer domain, respectively, are fused to both N-terminus and C-terminus of a starting rare-cutting endonuclease scaffold according to the invention.
  • both cleavase enhancer domain and nickase enhancer domain can be fused on an inactive rare-cutting endonuclease scaffold which serves as a scaffold for targeted DNA binding.
  • the present invention concerns a method for the creation of fusion proteins that consist of engineering a fusion protein between a rare-cutting endonuclease-derived scaffold and at least one enhancer domain wherein said enhancer domain enhances the efficiency of said chimeric rare-cutting endonuclease when fused to it.
  • a method to create a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence comprising the steps of:
  • said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold. In another preferred embodiment, said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold. In another embodiment, two enhancer domains are fused to both N-terminus part of said rare-cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived monomer scaffold of a homodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising two or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived monomer scaffold of a heterodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two or three or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived scaffold from a single-chain endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two enhancer domains.
  • said enhancer domain is catalytically active or not, providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a protein domain derived from an exonuclease.
  • said enhancer domain consists of a protein domain derived from an endonuclease. In another more preferred embodiment, said enhancer domain consists of a protein domain derived from a cleavase. In another more preferred embodiment, said enhancer domain consists of a protein domain derived from a nickase.
  • said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinPll, l-Basl, I- Bmol, l-Hmul, l-Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB, Endodeoxyribonuclease I (
  • said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112).
  • any combinations of two protein domains selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinPll, l-Basl, l-Bmol, l-Hmul, I- Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonu
  • l-Hmul catalytic domain can be fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold and ColE7 can be fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • ColE7 can be fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a catalytically active derivative of the protein domains listed above and in Table 1, providing functional and/or structural support to said rare- cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a catalytically inactive derivative of the protein domains listed above and in Table 1, providing structural support to said rare-cutting endonuclease-derived scaffold.
  • said rare-cutting endonuclease-derived scaffold is derived from a meganuclease.
  • said meganuclease comprises two identical monomers.
  • said meganuclease comprises two non-identical monomers.
  • said meganuclease is a single-chain meganuclease.
  • said rare-cutting endonuclease-derived scaffold is derived from the group consisting of !-Crel, a functional mutant of l-Crel, a variant of l-Crel or a derivative thereof.
  • rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 or residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or two or three or four or five or six or seven or eight or nine or ten further amino acid mutations.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and K82A mutation.
  • said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of l-Crel_NFSl (SEQ ID NO: 6); l-Crel_NFS2 (SEQ ID NO: 7); and l-Crel_CFSl (SEQ ID NO: 8).
  • said chimeric rare-cutting endonuciease can comprise at least one peptidic linker between said rare-cutting endonuclease-derived scaffold and said at least one enhancer domain.
  • said peptidic linker sequence is selected from the group consisting of 2012/032386
  • the peptidic linker that can link said enhancer domain to the rare-cutting endonuclease-derived scaffold according to the method of the present invention can be selected from the group consisting of NFS1 (SEQ ID NO: 98), NFS2 (SEQ ID NO: 99) and CFS1 (SEQ ID NO: 100).
  • NFS1 SEQ ID NO: 98
  • NFS2 SEQ ID NO: 99
  • CFS1 SEQ ID NO: 100
  • Enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, can also be a consequence of a fuctional support by said at least one enhancer domain.
  • said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a cleavase. In another more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nickase. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an exonuclease.
  • Cleavage by rare-cutting endonucleases usually generates cohesive ends, with 3' overhangs for LAGLIDADG meganucleases ⁇ Chevalier, 2001 #6 ⁇ and 5' overhangs for Zinc Finger Nucleases ⁇ Smith, 2000 #7 ⁇ . These ends, which result from hydrolysis of phosphodiester bonds, can be re-ligated in vivo by NHEJ in a seamless way (i.e a scarless re-ligation).
  • the restoration of a cleavable target sequence allows for a new cleavage event by the same endonuclease, and thus, a series of futile cycles of cleavage and re-ligation events can take place (Figure 4A).
  • enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention can be the hydrolysis of additional
  • nickase or cleavase activities to the existing cleavase activity of a rare-cutting endonuclease enhances the efficiency of the resulting chimeric rare-cutting endonuclease according to the invention, at a genomic locus of interest.
  • addition of two nickase activities on opposite strands (as depicted on Figure 4D) or of a new cleavase activity generating a second DSB ( Figure 4E) can result in a gap.
  • perfect re-ligation is not possible anymore, and one or several alternative repair outcomes such as imprecise NHEJ, Homologous Recombination or SSA for instance, can be stimulated.
  • the addition of a single nickase activity can result in a single strand gap, and suppress the cohesivity of the ends, which can also enhances the efficiency of the resulting chimeric rare-cutting endonuclease at a genomic locus of interest, according to the invention, via stimulation of one or several alternative repair outcomes mentioned above.
  • the method according to the invention enhances rare-cutting endonuclease efficiency for a DNA target sequence.
  • Enhancement of efficiency of a rare-cutting endonuclease according to the present invention can be a consequence of a structural support by said at least one enhancer domain.
  • said structural support enhances the binding of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as a non-limiting example.
  • said structural support by at least one enhancer domain enhances the existing catalytical activity of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as another non-limiting example.
  • said at least one enhancer domain enhances both the binding and the existing catalytical activity of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as another non-limiting example. All these non- limiting examples lead to a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target at a genomic locus of interest, according to the present invention.
  • the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention.
  • the addition of one nickase domain to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one nickase activity (depicted in figure 4B).
  • the addition of two nickase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two nickase activities (as depicted in figures 5C and 5D).
  • the addition of one cleavase domain to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one cleavase activity (figure 4E).
  • the addition of two cleavase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two cleavase activities.
  • enhancement of efficiency of a rare-cutting endonuclease refers to the increase in the detected level of said efficiency, against a target DNA sequence, of a second rare-cutting endonuclease in comparison to the activity of a first rare-cutting endonuclease against the same target DNA sequence.
  • Said second rare-cutting endonuclease can be an engineered rare-cutting endonuclease, i.e. a chimeric rare-cutting endonuclease according to the invention or not.
  • Said first rare-cutting endonuclease can be a wild-type rare-cutting endonuclease, a starting rare-cutting endonuclease, an engineered rare-cutting endonuclease i.e a chimeric rare-cutting endonuclease according to the invention or not, taken as a reference scaffold for enhancement in efficiency. Additional rounds of enhancement can be envisioned on a starting rare-cutting endonuclease scaffold.
  • Said enhancement of efficiency can be determined, as non-limiting example, by measuring the level of cleavage-induced recombination generated by said rare-cutting endonuclease or said chimeric rare-cutting endonuclease according to the present invention. Said level of cleavage-induced recombination can be measured by a cell-based recombination assay as described in the International PCT Application WO 2004/067736, as non-limiting example.
  • enhancement can be measured by
  • 33 of efficacy in cells is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays.
  • additional phosphodiesterase activities as described in Figure 4 could barely affect the cleavage profile, as detected by in vitro cleavage and separation of the cleavage products on an electrophoresis gel.
  • the DSB ends generated in this way could be more prone to induce detectable genomic rearrangements such as targeted mutagenesis (by imprecise NHEJ) or homologous recombination.
  • Said enhancement in efficiency of said rare-cutting endonuclease is at least a 5% enhancement compared to the starting scaffold, more preferably at least a 10% enhancement, again more preferably at least a 15% enhancement, again more preferably at least a 20% enhancement, again more preferably at least a 25% enhancement, again more preferably a 50% enhancement, again more preferably a enhancement greater than 50%.
  • the enhancement of efficiency of a rare-cutting endonuclease according to the method of the present invention can also be an enhancement in the efficiency for 2NN derivatives of a DNA target sequence by a rare-cutting endonuclease from the LAGLIDADG meganuclease subfamily.
  • 2NN derivatives of a DNA target sequence is intended variants of a given DNA target sequence having mutations in the four central nucleotides -2 to +2 as numbered in figure 2.
  • the method according to the invention enhances the efficiency for derivatives of the DNA target sequence C1221 having mutations in the four central nucleotides -2 to +2 as numbered in figure 2, i.e that do not have GTAC sequence at position -2 to +2 as numbered in figure 2.
  • the method according to the invention allows recombination to be initiated at 2NN derivatives that are not normally processed by a starting rare-cutting endonuclease scaffold from the LAGLIDADG meganuclease subfamily.
  • the method according to the invention allows the processing of 2NN derivatives that have -2 to +2 target sequences as listed in Table 4 and Table 5, non-originally cleaved by l-Crel.
  • the present invention also concerns the creation of functional single polypeptide fusion proteins for simple and efficient vectorization.
  • the present invention relates to chimeric rare-cutting endonucleases comprising at least an enhancer domain wherein said enhancer domain enhances the efficiency of said rare- cutting endonuclease when fused to it, thereby obtaining a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence compared to a corresponding rare-cutting endonuclease lacking said enhancer domain.
  • a chimeric rare-cutting endonuclease for a DNA target sequence comprising:
  • an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease for said DNA target sequence when fused to it;
  • said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold. In another preferred embodiment, said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold. In another embodiment, two enhancer domains are fused to both N-terminus part of said rare-cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived monomer scaffold of a homodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising two or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived monomer scaffold of a heterodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two or three or four enhancer domains.
  • said enhancer domain(s) are fused to N-terminus and/or C-terminus of a rare-cutting endonuclease- derived scaffold from a single-chain endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two enhancer domains.
  • said enhancer domain is catalytically active, providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a protein domain derived from an exonuclease.
  • said enhancer domain consists of a protein domain derived
  • said enhancer domain consists of a protein domain derived from a cleavase. In another more preferred embodiment, said enhancer domain consists of a protein domain derived from a nickase.
  • said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinPll, l-Basl, I- Bmol, l-Hmul, l-Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB, Endodeoxyribonuclease I (
  • said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112).
  • any combinations of two protein domains selected from the group consisting of Mmel, Colicin-E7 (CEA7_ECOLX), EndA, Endo I (ENDl_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinPll, l-Basl, l-Bmol, l-Hmul, I- Tevl, l-Tevll, l-Tevlll, l-Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonu
  • l-Hmul catalytic domain can be fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold and ColE7 can be fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.
  • said enhancer domain consists of a catalytically active derivative of the protein domains listed above
  • said enhancer domain consists of a catalytically inactive derivative of the protein domains listed above and in Table 1, providing structural support to said rare-cutting endonuclease-derived scaffold.
  • said rare-cutting endonuclease-derived scaffold is derived from a meganuclease.
  • said meganuclease comprises two identical monomers.
  • said meganuclease comprises two non-identical monomers.
  • said meganuclease is a single-chain meganuclease.
  • said rare-cutting endonuclease-derived scaffold is derived from the group consisting of l-Crel, a functional mutant of l-Crel, a variant of l-Crel or a derivative thereof.
  • rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1).
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or several amino acids substitutions.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 or residues 2 to 155 of wild-type l-Crel (SEQ ID NO: 1) and one or two or three or four or five or six or seven or eight or nine or ten further amino acid mutations.
  • rare-cutting endonuclease-derived scaffold of the present invention comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1) and K82A mutation.
  • said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of l-Crel_NFSl (SEQ ID NO: 6); l-Crel_NFS2 (SEQ ID NO: 7); and l-Crel_CFSl (SEQ ID NO: 8).
  • said chimeric rare-cutting endonuclease can optionally comprise at least one peptidic linker between said rare-cutting endonuclease-derived scaffold and said at least one enhancer domain.
  • said peptidic linker sequence is selected from
  • the peptidic linker that can link said enhancer domain to the rare-cutting endonuclease-derived scaffold according to the present invention can be selected from the group consisting of NFS1 (SEQ ID NO: 98), NFS2 (SEQ ID NO: 99) and CFS1 (SEQ ID NO: 100).
  • NFS1 SEQ ID NO: 98
  • NFS2 SEQ ID NO: 99
  • CFS1 SEQ ID NO: 100
  • Enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, can also be a consequence of a fuctional support by said at least one enhancer domain.
  • said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease.
  • said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a cleavase. In another more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nickase. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an exonuclease.
  • Cleavage by rare-cutting endonucleases usually generates cohesive ends, with 3' overhangs for LAGLIDADG meganucleases ⁇ Chevalier, 2001 #6 ⁇ and 5' overhangs for Zinc Finger Nucleases ⁇ Smith, 2000 #7 ⁇ . These ends, which result from hydrolysis of phosphodiester bonds, can be re-ligated in vivo by NHEJ in a seamless way (i.e a scarless re-ligation).
  • the restoration of a cleavable target sequence allows for a new cleavage event by the same endonuclease, and thus, a series of futile cycles of cleavage and re-ligation events can take place (Figure 4A).
  • enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention can be the hydrolysis of additional
  • nickase or cleavase activities to the existing cleavase activity of a rare-cutting endonuclease enhances the efficiency of the resulting chimeric rare-cutting endonuclease according to the invention, at a genomic locus of interest.
  • addition of two nickase activities on opposite strands (as depicted on Figure 4D) or of a new cleavase activity generating a second DSB ( Figure 4E) can result in a gap.
  • perfect re-ligation is not possible anymore, and one or several alternative repair outcomes such as imprecise NHEJ, Homologous Recombination or SSA for instance, can be stimulated.
  • the addition of a single nickase activity can result in a single strand gap, and suppress the cohesivity of the ends, which can also enhances the efficiency of the resulting chimeric rare-cutting endonuclease at a genomic locus of interest, according to the invention, via stimulation of one or several alternative repair outcomes mentioned above.
  • chimeric rare-cutting endonucleases according to the invention enhances rare-cutting endonuclease efficiency for a DNA target sequence. Enhancement of efficiency of a rare-cutting endonuclease according to the present invention can be a consequence of a structural support by said at least one enhancer domain.
  • said structural support enhances the binding of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as a non-limiting example.
  • said structural support by at least one enhancer domain enhances the existing catalytical activity of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as another non-limiting example.
  • said at least one enhancer domain enhances both the binding and the existing catalytical activity of a chimeric rare-cutting endonuclease according to the invention for a DNA target sequence compared to the binding of a starting rare-cutting endonuclease for the same DNA target sequence, as another non- limiting example. All these non-limiting examples lead to a chimeric rare-cutting endonuclease with
  • the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention.
  • the addition of one nickase domain to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one nickase activity (depicted in figure 4B).
  • the addition of two nickase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two nickase activities (as depicted in figures 5C and 5D).
  • the addition of one cleavase domain to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and one cleavase activity (figure 4E).
  • the addition of two cleavase domains to an active rare-cutting endonuclease scaffold derived from a meganuclease results in a chimeric rare-cutting endonuclease with one meganuclease activity and two cleavase activities.
  • enhancement of efficiency of a rare-cutting endonuclease refers to the increase in the detected level of said efficiency, against a target DNA sequence, of a second rare-cutting endonuclease in comparison to the activity of a first rare-cutting endonuclease against the same target DNA sequence.
  • Said second rare-cutting endonuclease can be an engineered rare-cutting endonuclease, i.e. a chimeric rare-cutting endonuclease according to the invention or not.
  • Said first rare-cutting endonuclease can be a wild-type rare-cutting endonuclease, a starting rare-cutting endonuclease, an engineered rare-cutting endonuclease i.e a chimeric rare-cutting endonuclease according to the invention or not, taken as a reference scaffold for enhancement in efficiency. Additional rounds of enhancement can be envisioned on a starting rare-cutting endonuclease scaffold.
  • Said enhancement of efficiency can be determined, as non-limiting example, by measuring the level of cleavage-induced recombination generated by said rare-cutting endonuclease or said chimeric rare-cutting endonuclease according to the present invention. Said level of cleavage-induced
  • enhancement of efficacy in cells can be, but is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays.
  • additional phosphodiesterase activities as described in Figure 4 could barely affect the cleavage profile, as detected by in vitro cleavage and separation of the cleavage products on an electrophoresis gel.
  • the DSB ends generated in this way could be more prone to induce detectable genomic Rearrangements such as targeted mutagenesis (by imprecise NHEJ) or homologous recombination.
  • Said enhancement in efficiency of said rare-cutting endonuclease is at least a 5% enhancement compared to the starting scaffold, more preferably at least a 10% enhancement, again more preferably at least a 15% enhancement, again more preferably at least a 20% enhancement, again more preferably at least a 25% enhancement, again more preferably a 50% enhancement, again more preferably a enhancement greater than 50%.
  • the enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention can also be an enhancement in the efficiency for 2NN derivatives of a DNA target sequence by a rare-cutting endonuclease from the LAGLIDADG meganuclease subfamily.
  • 2NN derivatives of a DNA target sequence is intended variants of a given DNA target sequence having mutations in the four central nucleotides -2 to +2 as numbered in figure 2.
  • a chimeric rare-cutting endonuclease according to the invention can enhance the efficiency for derivatives of the DNA target sequence C1221 having mutations in the four central nucleotides -2 to +2 as numbered in figure 2, i.e that do not have GTAC sequence at position -2 to +2 as numbered in figure 2.
  • the chimeric rare-cutting endonuclease according to the invention can allow the processing of 2NN derivatives that are not efficiently processed by a starting rare-cutting endonuclease scaffold from the LAGLIDADG meganuclease subfamily.
  • the chimeric rare-cutting endonuclease according to the invention allows the induction of recombination by processing 2NN derivatives that have -2 to +2 target sequences as listed in Table 4 and Table 5, non-originally cleaved by l-Crel.
  • said chimeric rare-cutting endonuclease is selected from the group consisting of hHmuCre_D0101 (SEQ ID NO: 9), hHmuCre_D0201 (SEQ ID NO: 105), hHmuCre_D0301 (SEQ ID NO: 106), hHmuCre_D0401 (SEQ ID NO: 107), hHmuCre_D0102 (SEQ ID NO: 9), hHmuCre_D0201 (SEQ ID NO: 105), hHmuCre_D0301 (SEQ ID NO: 106), hHmuCre_D0401 (SEQ ID NO: 107), hHmuCre_D0102 (SEQ ID NO: 9), hHmuCre_D0201 (SEQ ID NO: 105), hHmuCre_D0301 (SEQ ID NO: 106), hHmuCre_D0401 (SEQ
  • the present invention relates to a method for treatment of a genetic disease caused by a mutation in a specific single double-stranded DNA target sequence in a gene, comprising administering to a subject in need thereof an effective amount of a chimeric rare-cutting endonuclease according to the present invention.
  • the present invention relates to a method for inserting a transgene into a specific single double-stranded DNA target sequence of a genomic locus of a cell, tissue or non-human animal wherein at least one chimeric rare-cutting endonuclease of the present invention is introduced in said cell, tissue or non-human animal.
  • the chimeric rare-cutting endonuclease of the present invention 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.
  • 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.
  • Vectors comprising targeting DNA and/or nucleic acid encoding a chimeric rare-cutting endonuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). Chimeric rare-cutting endonucleases 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”).
  • the chimeric rare-cutting endonuclease of the present invention 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.
  • the chimeric rare-cutting endonuclease is substantially free of N-formyl methionine.
  • Another way to avoid unwanted immunological reactions is to conjugate chimeric rare-cutting endonuclease 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
  • the present invention relates to recombinant polynucleotides encoding the chimeric rare-cutting endonucleases of the present invention, specific vectors (polynucleotidic or not) encoding and/or vectorizing them, compositions and/or kits comprising them, all of them beign used or part of a whole to implement methods of the present invention for enhancing rare-cutting endonuclease cleavage activity at a genomic locus of interest in a cell.
  • - Amino acid substitution means the replacement of one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a peptide sequence is an amino acid substitution.
  • Efficiency of a rare-cutting endonuclease is the property for said rare-cutting endonuclease of producing a desired event.
  • This desired event can be for example Homologous gene targeting, targeted mutagenesis, or sequence removal or excision.
  • the efficiency of the desired event depends on several parameters, including the specific activity of the nuclease and the repair pathway resulting in the desired event (efficacy of homologous repair for gene targeting, efficacy and outcome of NHEJ pathways for targeted mutagenesis).
  • Efficiency of a rare cutting endonuclease for a locus is intended to mean its abiliy to produce a desired event at this locus.
  • Efficiency of a rare cutting endonuclease for a target is intended to mean its abiliy to produce a desired event as a consequence of cleavage of this target.
  • Enhancement of efficiency of a rare-cutting endonuclease by creating a chimeric rare- cutting endonuclease with enhanced efficiency according to the present invention is measurable comparatively to the efficiency of the starting rare-cutting endonuclease scaffold in the same conditions.
  • Enhancement of efficiency of a chimeric rare-cutting endonuclease can be the consequence of an enhancement of an individual structural parameter or an enhancement of a combination of several individual structural parameters consecutive to the addition of an enhancer domain according to the present invention.
  • Such parameters can be as non-limiting examples, binding affinity or capacity of binding, cleavage activity, turn-over of one enzyme (Kcat), Kcat/Km (incorporating the different rate constants of an enzymatic reaction).
  • Enhancement of efficiency of a chimeric rare-cutting endonuclease can also be a functional consequence to the addition of an enhancer domain according to the present invention.
  • Said additional enhancer domain can provide additional phosphodiester bonds hydrolysis, via cleavase or nickase domains as non-limiting examples, allowing to favour or stimulate one or several alternative repair outcomes.
  • enhancement of efficiency can also be an enhancement in th eefficiency for 2NN derivatives of DNA target sequence originally targeted by the parent rare-cutting endonuclease.
  • an enhancer domain can provide additional phosphodiester bonds hydrolysis, via cleavase or nickase domains as non-limiting examples, allowing to favour or stimulate one or several alternative repair outcomes.
  • enhancement of efficiency can also be an enhancement in th eefficiency for 2NN derivatives of DNA target sequence originally targeted by the parent rare-cutting endonuclease.
  • an enhancer domain can provide additional phosphodiester bonds hydrolysis, via cleavase or nick
  • enhancement of the efficiency of chimeric rare-cutting endonucleases derived from LAGLIDADG meganuclease subfamily can be the capacity to process 2NN derivatives that are not cleaved in a detectable way by the parent rare-cutting endonuclease.
  • enhancement of efficiency of a rare- cutting endonuclease according to the present invention can be measured from an application point of view.
  • Chimeric rare-cutting endonucleases according to the present invention being usable for various genome engineering applications, said chimeric rare-cutting endonucleases according to the invention can have an enhanced efficiency for NHEJ, HR, SSA, as non-limiting examples.
  • Said chimeric rare-cutting endonucleases according to the invention can also have an enhanced efficiency for a precise genome engineering such as gene targeting, targeted DNA cleavage or targeted gene repression as non-limiting examples.
  • Enhanced/increased/improved cleavage activity or binding refers to an increase in the detected level of an endonuclease, a rare-cutting endonuclease or a chimeric rare-cutting endonuclease cleavage activity or binding, see below, against a target DNA sequence or DNA target sequence by a second endonuclease, rare-cutting endonuclease or chimeric rare-cutting endonuclease in comparison to the cleavage activity or binding of a first endonuclease or a wild-type endonuclease (non chimeric) or a first rare-cutting endonuclease or a first chimeric rare-cutting endonuclease against said target DNA sequence.
  • the second endonuclease or rare-cutting endonuclease or chimeric rare-cutting endonuclease can be a variant of the first one and can comprise one or more substituted amino acid residues in comparison to the first endonuclease or rare-cutting endonuclease or chimeric rare-cutting endonuclease or additional peptidic sequences or domains. Parameters in this definition are included in the method for enhancing rare-cutting endonuclease efficiency according to the present invention.
  • nucleosides 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.
  • 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
  • n represents g, a, t or c.
  • meganuclease is intended a rare-cutting endonuclease subtype having a double- stranded DNA target sequence greater than 12 bp.
  • Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomeric enzyme comprising the two domains on a single polypeptide.
  • “meganuclease domain” is intended the region which interacts with one half of the DNA target of a meganuclease and is able to associate with the other domain of the same meganuclease
  • the starting endonuclease, rare-cutting endonuclease, chimeric rare-cutting endonuclease or meganuclease also the starting scaffold with at least a different amino acid or additional peptidic sequences or domains.
  • peptide linker by "peptide linker", “peptidic linker” or “peptide spacer” it is intended to mean a peptide sequence which allows the connection of different monomers in a fusion protein and the adoption of the correct conformation for said fusion protein activity and which does not alter the specificity of either of the monomers for their targets.
  • Peptide linkers can be of various sizes, from 3 amino acids to 50 amino acids as a non limiting indicative range. Peptide linkers can also be structured or unstructured.
  • -variants or mutants of the structural elements of the invention including a rare-cutting endonuclease-derived scaffold, an enhancer domain, or a peptidic linker are contemplated.
  • these will be at least 80, 90, 95, 98, or 99% identical to a known element, such as those described herein, or have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions or substitutions compared to a corresponding element described herein.
  • one cell type related to the chosen cell type or organism is intended a cell type or an organism sharing characteristics with said chosen cell type or said chosen organism; this cell type or organism related to the chosen cell type or organism, can be derived from said chosen cell type or organism or not.
  • targeting DNA construct/minimal repair matrix/repair matrix it is intended to mean a DNA construct comprising a first and second portions that are homologous to regions 5' and 3' of the DNA target in situ.
  • the DNA construct also comprises a third portion positioned between the first and second portion which comprise some homology with the corresponding DNA sequence in situ or alternatively comprise no homology with the regions 5' and 3' of the DNA target in situ.
  • a homologous recombination event is stimulated between the genome containing the targeted gene comprised in the locus of interest and the repair matrix, wherein the
  • a functional variant is intended a catalytically active variant of a protein, such variant can have additional properties compared to its parent protein.
  • a functional variant of a meganuclease can be able to cleave additional DNA target sequences that are not cleaved by the parent meganuclease.
  • a meganuclease variant which is created from a parent meganuclease and hence the peptide sequence of the meganuclease variant is related to (primary sequence level) but derived from (mutations) the peptide sequence of the parent meganuclease.
  • mutations encompass deletions or insertions of several amino acid residues; as non-limiting example, a truncated variant of an l-Crel meganuclease is considered as a scaffold derived from l-Crel meganuclease.
  • This expression can be applied to an endonuclease, a rare-cutting endonuclease or a chimeric rare-cutting endonuclease.
  • l-Crel is intended the wild-type l-Crel having the sequence of pdb accession code lg9y, corresponding to the sequence SEQ ID NO: 1 in the sequence listing.
  • Application l-Cre ⁇ variants described can comprise an additional Alanine after the first Methionine of the wild type l-Cre ⁇ sequence (SEQ ID NO: 1). These variants may also comprise two additional Alanine residues and an Aspartic Acid residue after the final Proline of the wild type l-Crei sequence as shown in SEQ ID NO: 4.
  • l-Crel site is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by l-Crel.
  • l-Crel sites include the wild-type non-palindromic l-Crel homing site and the derived palindromic sequences such as the sequence 5'- t.nCna.joa.ga.ga.yC.eg.st ⁇ c.Bg ⁇ t. ia +1 c +2 g + 3a + C + 5g +6 t + 7t + 8t +9 t + i 0 g + 1 ia +12 (SEQ ID NO: 2), also called C1221.
  • domain or “core domain” is intended the "LAG LIDADG homing endonuclease core domain” which is the characteristic ⁇ 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 ( ⁇ , ⁇ folded in an anti-parallel 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.
  • the dimeric homing the dimeric homing
  • LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.
  • subdomain is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half-site.
  • beta-hairpin is intended two consecutive beta-strands of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease core domain ( ⁇ 2 0 ⁇ ⁇ 3 ⁇ 4 ) which are connected by a loop or a turn,
  • single-chain meganuclease is intended a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic linker or 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.
  • This definition can be enlarged to "single-chain endonuclease” or “single-chain endonuclease derivative” that qualify two endonuclease domains or core domains from non-LAGLIDADG endonucleases linked by a peptidic linker or peptidic spacer.
  • cleavage site is intended a double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease such as l-Crel, or a variant, or a single-chain chimeric meganuclease derived from l-Crel.
  • Said DNA target sequence is qualified as "cleavable" by an endonuclease, rare-cutting endonuclease, chimeric rare- cutting endonuclease or meganuclease when recognized within a genomic sequence and known to correspond to the DNA target sequence of a given endonuclease, rare-cutting endonuclease, chimeric rare-cutting endonuclease or meganuclease or a variant of such endonuclease, rare-cutting endonuclease, chimeric rare-cutting endonuclease or meganuclease.
  • a distinct DNA location preferably a genomic location but also a portion of genetic material that can exist independently to the main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria or chloroplasts as non-limiting examples, at which at least a double-strand break (cleavage) or a single strand break (nick) is to be induced by the meganuclease, endonuclease, rare-cutting endonuclease or chimeric rare-cutting endonuclease.
  • the DNA target is defined by the 5' to 3' sequence of one strand of the double-stranded polynucleotide, as indicate above for C1221 (SEQ ID NO: 2, figure 2). Cleavage of the DNA target occurs at the nucleotides at positions +2 and -2, respectively for the
  • Endonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or NA molecule, preferably a DNA molecule.
  • Endonucleases can be classified as rare-cutting endonucleases when having typically a polynucleotide recognition site greater than 12base pairs (bp) in length, more preferably of 14-45 bp.
  • Rare-cutting endonucleases significantly increase HR by inducing DNA double-strand breaks (DSBs) at a defined locus (Rouet, Smih et al. 1994; Rouet, Smih et al.
  • Rare-cutting endonucleases can for example be a homing endonuclease (Paques and Duchateau 2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus and Carroll 2005) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005 ; Arimondo, Thomas et al. 2006; Simon, Cannata et al. 2008).
  • a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence.
  • Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention.
  • Rare-cutting endonucleases can also be for example TALENs, a new class of chimeric nucleases using a Fokl catalytic domain and a DNA binding domain derived from Transcription Activator Like Effector (TALE), a family of proteins used in the infection process by plant pathogens of the Xanthomonas genus (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2010).
  • TALE Transcription Activator Like Effector
  • the functional layout of a Fokl-based TALE-nuclease (TALEN) is essentially that of a ZFN, with the Zinc-finger DNA binding domain being replaced by the TALE domain.
  • DNA cleavage by a TALEN requires two DNA recognition regions flanking an unspecific central region.
  • Rare-cutting endonucleases encompassed in the present invention can also be derived from
  • Rare-cutting endonuclease can be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the art (Stodd
  • 49 12 032386 to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease.
  • HEs Homing Endonucleases
  • proteins families Cholier and Stoddard 2001.
  • 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.
  • LAGLIDADG The LAGLIDADG family, named after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Many structures are now available. Whereas most proteins from this family are monomeric and display two LAGLIDADG motifs, a few have only one motif, and thus dimerize to cleave palindromic or pseudo-palindromic target sequences.
  • LAGLIDADG peptide is the only conserved region among members of the family, these proteins share a very similar architecture.
  • the catalytic core is flanked by two DNA- binding domains with a perfect two-fold symmetry for homodimers such as l-Crel (Chevalier, Monnat et al. 2001), l- sol (Chevalier, Turmel et al. 2003) and l-Ceul (Spiegel, Chevalier et al. 2006) and with a pseudo symmetry for monomers such as l-Scel (Moure, Gimble et al. 2003), l-Dmol (Silva, Dalgaard et al.
  • residues 28 to 40 and 44 to 77 of l-Crel were shown to form two partially separable functional subdomains, able to bind distinct parts of a homing endonuclease target half- site (Smith, Grizot et al. 2006); International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)).
  • the combination of the two former steps allows a larger combinatorial approach, involving four different subdomains.
  • the different subdomains can be modified separately and combined to obtain an entirely redesigned meganuclease variant (heterodimer or single-chain molecule) with chosen specificity.
  • couples of novel meganucleases are combined in new molecules
  • half-meganucleases cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such "half-meganucleases” can result in a heterodimeric species cleaving the target of interest.
  • Examples of such endonuclease include l-Sce I, l-Chu I, l-Cre I, l-Csm I, Pl-Sce I, Pl-Tli I, Pl-Mtu I, l-Ceu I, l-Sce II, l-Sce III, HO, Pl-Civ I, Pl-Ctr I, Pl-Aae I, Pl-Bsu I, Pl-Dha I, Pl-Dra I, Pl-Mav I, Pl-Mch I, Pl-Mfu I, Pl-Mfl I, Pl-Mga I, Pl-Mgo I, Pl-Min I, Pl-Mka I, Pl-Mle I, Pl-Mma I, Pl-Msh I, Pl-Msm I, Pl-Mth I, Pl-Mtu I, Pl-Mxe I, Pl-Npu I, Pl-Pfu I, Pl-Rma I, Pl-Spb I, Pl-Ss
  • a homing endonuclease can be a LAGLIDADG endonuclease such as ⁇ -Scel, ⁇ -Crel, ⁇ -Ceul, I- Msol, and ⁇ -Dmol.
  • Said LAGLIDADG endonuclease can be l-Sce I, a member of the family that contains two LAGLIDADG motifs and functions as a monomer, its molecular mass being approximately twice the mass of other family members like l-Crel which contains only one LAGLIDADG motif and functions as homodimers.
  • Meganucleases, endonucleases or rare-cutting endonucleases mentioned in the present application encompass both wild-type (naturally-occurring) and variant meganucleases, endonucleases or rare-cutting endonucleases.
  • Meganucleases, endonucleases or rare-cutting endonucleases according to the invention can be a "variant" meganuclease, endonuclease or rare- cutting endonuclease, i.e.
  • a meganuclease, endonuclease or rare-cutting endonuclease that does not naturally exist in nature and that is obtained by genetic engineering or by random mutagenesis, i.e. an engineered meganuclease, endonuclease or rare-cutting endonuclease.
  • This variant meganuclease, endonuclease or rare-cutting endonuclease can for example be obtained by substitution of at least one residue in the amino acid sequence of a wild-type, naturally-occurring, meganuclease, endonuclease or rare-cutting endonuclease with a different amino acid.
  • substitution(s) can for example be introduced by site-directed mutagenesis and/or by random mutagenesis.
  • such variant meganucleases, endonucleases or rare-cutting endonucleases remain functional, i.e. they retain the capacity of recognizing (binding
  • Meganuclease part, endonuclease part or rare-cutting endonuclease part in a chimeric rare- cutting endonuclease according to the present invention can only retain the capacity of recognizing a target sequence, in particular case, the capacity of cleaving a target sequence being provided by an enhancer domain according to the present invention.
  • the variant meganuclease, endonuclease or rare-cutting endonuclease according to the invention cleaves a target sequence that is different from the target sequence of the corresponding wild-type meganuclease, endonuclease or rare-cutting endonuclease.
  • Methods for obtaining such variant endonucleases with novel specificities are well-known in the art.
  • Endonucleases variants may be homodimers (meganuclease comprising two identical monomers) or heterodimers (meganuclease comprising two non-identical monomers). It is understood that the scope of the present invention also encompasses endonuclease variants per se, including heterodimers (WO2006097854), obligate heterodimers (WO2008093249) and single chain meganucleases (WO03078619 and WO2009095793) as non limiting examples, able to cleave one target of interest in a polynucleotide sequence or in a genome and derivatives of these variants according to the present invention, such as chimeric rare-cutting endonucleases derived from these variants.
  • the invention also encompasses hybrid variant per se composed of two monomers from different origins (WO03078619).
  • Meganucleases, endonucleases or rare-cutting endonucleases with novel specificities can be used in the method according to the present invention for gene targeting and thereby integrating a transgene of interest into a genome at a predetermined location.
  • parent meganuclease it is intended to mean a wild type meganuclease or a variant of such a wild type meganuclease with identical properties or alternatively a meganuclease with some altered characteristic in comparison to a wild type version of the same meganuclease.
  • This expression can be applied to an endonuclease, a rare-cutting endonuclease or a chimeric rare- cutting endonuclease.
  • delivery vector or “ delivery vectors” is intended any delivery vector which can be used in the present invention to put into cell contact ( i.e “contacting") or deliver inside cells or subcellular compartments agents/chemicals and molecules (proteins or nucleic acids) needed in the present invention, such as meganucleases, endonucleases, rare-cutting endonucleases or chimeric rare-cutting endonucleases or plasmidic vectors encoding said meganucleases, endonucleases, rare-cutting endonucleases or chimeric rare-cutting endonucleases, or repair matrix per se or encoded in a plasmidic vector for a gene engineering application for instance.
  • liposomal delivery vectors viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other appropriate transfer vectors.
  • delivery vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, peptides developed by Diatos. In these cases, delivery vectors are molecule carriers.
  • delivery vector or “delivery vectors” is also intended delivery methods to perform transfection.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a “vector” in the present invention includes, but is not limited to, a viral vector, a plasmid (i.e a plasmidic vector), a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids.
  • Preferred vectors are those capable of autonomous replication (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. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (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.
  • orthomyxovirus e. g., influenza virus
  • rhabdovirus e. g., rabies and vesicular stomatitis virus
  • paramyxovirus e. g. measles and Sendai
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • 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).
  • lentiviral vector HIV-Based lentiviral vectors that are very promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types.
  • Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells.
  • lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface.
  • the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex.
  • the product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells.
  • integrated lentiviral vectors or LV
  • integrated lentiviral vectors or LV
  • non integrative lentiviral vectors or NILV is meant efficient gene delivery vectors that do not integrate the genome of a target cell through the action of the virus integrase.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.
  • a vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a viral vector, a plasmid, a NA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • 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 5. cerevisiae; tetracyclin, rifampicin or ampicillin resistance in E. coli.
  • selectable markers for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine
  • said vectors are expression vectors, wherein a sequence encoding a polypeptide of interest is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said polypeptide. 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, a RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer or silencer elements. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Suitable promoters include tissue specific and/or inducible promoters.
  • 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-p-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature.
  • tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), ct-antitrypsin protease, human surfactant (SP) A and B proteins, ⁇ - casein and acidic whey protein genes.
  • Inducible promoters may be induced by pathogens or stress, more preferably by stress like cold, heat, UV light, or high ionic concentrations (reviewed in Potenza C et al. 2004, In vitro Cell Dev Biol 40:1-22). Inducible promoter may be induced by chemicals (reviewed in (Moore, Samalova et al. 2006); (Padidam 2003); (Wang, Zhou et al. 2003); (Zuo and Chua 2000).
  • Delivery vectors and vectors can be associated or combined with any cellular permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques.
  • cell or cells any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.
  • primary cell or “primary cells” are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. These cells thus represent a more valuable model to the in vivo state they refer to.
  • fungus refers to a fungal, plant or animal cell or a cell line derived from the organisms listed below and established for in vitro culture. More preferably, the fungus is of the genus Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces or Pichia; More preferably, the fungus is of the species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum, Penicillium citrinum, Acremonium Chrysogenum, Trichoderma reesei, Mortierella alpine, Chrysosporium lucknowense, Kluyveromyces lactis, Pichia pastoris or Pichia ciferrii.
  • the plant is of the genus Arabidospis, Nicotiana, Solanum, lactuca, Brassica, Oryza, Asparagus, Pisum, Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis, Citrus, Sorghum; More preferably, the plant is of the species Arabidospis thaliana, Nicotiana tabaccum, Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Solanum esculentum, Lactuca saliva, Brassica napus, Brassica oleracea, Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus officinalis, Pisum sativum, Medicago sativa, zea mays, Hordeum vulgare, Secale cereal, Triticum aestivum, Triticum durum, Capsicum sativus, Cu
  • the animal cell is of the genus Homo, Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus, Meleagris, Drosophila, Caenorhabditis; more preferably, the animal cell is of the species Homo sapiens, Rattus norvegicus, Mus musculus, Sus scrofa, Bos
  • the cell can be a plant cell, a mammalian cell, a fish cell, an insect cell or cell lines derived from these organisms for in vitro cultures or primary cells taken directly from living tissue and established for in vitro culture.
  • cell lines can be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; M C5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
  • All these cell lines can be modified by the method of the present invention to provide cell line models to produce, express, quantify, detect, study a gene or a protein of interest; these models can also be used to screen biologically active molecules of interest in research and production and various fields such as chemical, biofuels, therapeutics and agronomy as non-limiting examples.
  • Adoptive immunotherapy using genetically engineered T cells is a promising approach for the treatment of malignancies and infectious diseases. Most current approaches rely on gene transfer by random integration of an appropriate T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR).
  • TCR T Cell Receptor
  • CAR Chimeric Antigen Receptor
  • Targeted approach using rare-cutting endonucleases such as meganucleases is an efficient and safe alternative method to transfer genes into T cells and generate genetically engineered T cells.
  • homologous is intended a sequence with enough identity to another one to lead to 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 setting.
  • mutant is intended the substitution, deletion, insertion 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.
  • double-strand break-induced mutagenesis (DSB-induced mutagenesis) and “targeted mutagenesis” refer to a mutagenesis event consecutive to an NHEJ event following an endonuclease-induced DSB, leading to insertion/deletion at the cleavage site of an endonuclease
  • gene is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein.
  • a gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region.
  • the gene may further comprise a terminator, enhancers and/or silencers.
  • transgene refers to a sequence encoding a polypeptide.
  • the polypeptide encoded by the transgene is either not expressed, or expressed but not biologically active, in the cell, tissue or individual in which the transgene is inserted.
  • the transgene encodes a therapeutic polypeptide useful for the treatment of an individual.
  • gene of interest refers to any nucleotide sequence encoding a known or putative gene product.
  • locus is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome.
  • locus usually refers to the specific physical location of an endonuclease's target sequence on a chromosome.
  • locus which comprises a target sequence that is recognized and cleaved by an endonuclease according to the invention, is referred to as “locus according to the invention”.
  • locus according to the invention is referred to as “locus according to the invention”.
  • the expression "genomic locus of interest” is used to qualify a nucleic acid sequence in a genome that can be a putative target for a double-strand break according to the invention.
  • genomic locus of interest is intended a native nucleic acid sequence in a genome, i.e. a sequence or allelic variations of this sequence that is naturally present at this genomic locus. It is understood that the considered genomic locus of interest of the present invention can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e. in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria or chloroplasts as non-limiting examples.
  • loss of genetic information is understood the elimination or addition of at least one given DNA fragment (at least one nucleotide) or sequence, bordering the recognition sites of the endonucleases, rare-cutting endonucleases or chimeric rare-cutting endonucleases of the present invention and leading to a change of the original sequence around said endonuclease-, rare-cutting endonuclease- or chimeric rare-cutting endonuclease-cutting sites, within the genomic locus of interest.
  • This loss of genetic information can be, as a non-limiting example, the elimination
  • this loss of genetic information can also be an excision of a single-strand of DNA spanning the starting rare-cutting endonuclease scaffold DNA binding region of a chimeric rare-cutting endonuclease according to the present invention when both one cleavase enhancer domain and one nickase enhancer domain, respectively, are part of said chimeric rare-cutting endonuclease.
  • the starting rare- cutting endonuclease scaffold DNA binding region can be catalytically active or not and the "loss of genetic information" corresponds to the elimination of an intervening DNA fragment between both cleavase and nickase cleavage sites of said chimeric rare-cutting endonuclease.
  • two nearby DNA double strand breaks within the genomic locus of interest, is meant two endonucleases cutting sites distant at between 12 bp and 1000 bp.
  • fusion protein is intended the result of a well-known process in the art consisting in the joining or merging of two or more genes which originally encode for separate proteins or part of them, the translation of said "fusion gene” resulting in a single polypeptide, said "fusion protein” with functional properties derived from each of the original proteins.
  • Imprecise NHEJ is intended the re-ligation of nucleic acid ends generated by a DSB, with insertions or deletions of nucleotides. Imprecise NHEJ is an outcome and not a repair pathway and can result from different NHEJ pathways (Ku dependent or Ku independent as non-limiting examples).
  • chimeric rare-cutting endonuclease any fusion protein comprising a rare- cutting endonuclease. Said rare-cutting endonuclease might be at the N- terminus part of said chimeric rare-cutting endonuclease; at the opposite, said rare-cutting endonuclease might be at the C- terminus part of said chimeric rare-cutting endonuclease.
  • a “chimeric rare-cutting endonuclease” according to the present invention which comprises two catalytic domains can be described as "bi- functional" or as "bi-functional meganuclease".
  • chimeric rare-cutting endonuclease which comprises more than two catalytic domains can be described as “multi- functional” or as “multi-functional meganuclease".
  • chimeric rare-cutting endonucleases according to the present invention can be a fusion protein between a rare-cutting endonuclease and one catalytic domain; chimeric rare-cutting endonucleases according to the present invention can also be a fusion protein between a rare-cutting endonuclease and two catalytic domains.
  • the rare-cutting endonuclease part of chimeric rare-
  • 59 cutting endonucleases according to the present invention can be a meganuclease comprising two identical monomers, two non-identical monomers, or a single chain meganuclease.
  • the rare-cutting endonuclease part of chimeric rare-cutting endonucleases according to the present invention can also be the DNA-binding domain of an inactive rare-cutting endonuclease.
  • chimeric rare-cutting endonucleases according to the present invention can be derived from a TALE-nuclease (TALEN), i. e.
  • a fusion between a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one or two catalytic domains are encompassed mutants, variants or derivatives of meganucleases (including monomers and single chain meganucleases), endonucleases, rare-cutting endonucleases or chimeric rare-cutting endonucleases.
  • TALE Transcription Activator Like Effector
  • hancer domain(s) or “enhancer(s)” are meant protein domains that provide functional and/or structural support to a protein scaffold, a rare-cutting endonuclease-derived scaffold as a non-limiting example, therefore allowing an enhancement in cleavage efficiency of the resulting fusion protein, i.e a chimeric rare-cutting endonuclease, relative to the cleavage efficiency of the wild-type rare-cutting endonuclease or starting rare-endonuclease scaffold.
  • a particular domain is an enhancer domain when it provides at least a 5% enhancement in efficiency of the starting scaffold, more preferably 10 %, again more preferably 15 %, again more preferably 20 %, again more preferably 25 %, again more preferably 50%, again more preferably greater than 50%.
  • enhancer domains are given in Table 1.
  • Enhancer domain(s) according to the present invention can be fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived monomer scaffold of a homodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising two or four enhancer domains.
  • Enhancer domain(s) according to the present invention can also be fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived monomer scaffold of a heterodimeric endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two or three or four enhancer domains.
  • Enhancer domain(s) according to the present invention can also be fused to N-terminus and/or C-terminus of a rare-cutting endonuclease-derived scaffold of a single-chain endonuclease, resulting in a chimeric rare-cutting endonuclease comprising one or two enhancer domains.
  • catalytic domain is intended the protein domain or module of an enzyme containing the active site of said enzyme; by active site is intended the part of said enzyme at which catalysis of the substrate occurs.
  • Enzymes, but also their catalytic domains, are classified and named according to the reaction they catalyze.
  • the Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze
  • any catalytic domain can be fused to a rare-cutting endonuclease scaffold or starting rare-endonuclease scaffold or starting scaffold to generate a chimeric rare-cutting endonuclease.
  • said catalytic domain can be an enhancer domain according to the present invention. If catalytically active, said enhancer domain can provide functional and/or structural support to rare-cutting endonuclease scaffold when fused to it. If catalytically inactive, said enhancer domain provides structural support to rare-cutting endonuclease scaffold when fused to it.
  • nuclease catalytic domain is intended the protein domain comprising the active site of an endonuclease or an exonuclease enzyme.
  • Such nuclease catalytic domain can be, for instance, a “cleavase domain” or a “nickase domain”.
  • cleavase domain is intended a protein domain whose catalytic activity generates a Double Strand Break (DSB) in a DNA target.
  • nickase domain is intended a protein domain whose catalytic activity generates a single strand break in a DNA target sequence.
  • Non-limiting examples of such catalytic domains are given in Table 1 with a GenBank or NCBI or UniProtKB/Swiss-Prot number as a reference.
  • Protein-fusion scaffolds were designed based on a truncated form of wild-type l-Crel (SEQ ID NO: 1), l-Crel_X (SEQ ID NO: 5) and three different linker polypeptides [NFSl (SEQ ID NO: 98), NFS2 (SEQ ID NO: 99) and CFS1 (SEQ ID NO: 100)] fused to either the N- or C-terminus of the protein. Structure models were generated in all cases, with the goal of designing a "baseline" fusion linker that would traverse the l-Crel parent scaffold surface with little to no effect on its DNA binding or cleavage activities.
  • the polypeptide spanning residues 2 to 153 of I- Crel was used, with a K82A mutation to allow for linker placement.
  • the C-terminal fusion scaffold contains residues 2 to 155 of wild-type l-Crel.
  • the "free" end of the linker i.e. onto which a polypeptide can be linked
  • l-Crel_CFSl SEQ ID NO: 8
  • yeast assay previously described in International PCT Applications WO 2004/067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006) and found to have activity similar to that of wild-type l-Crel.
  • Wild-type l-Hmul functions as a monomeric nickase to generate a strand-specific nick in its target DNA [REF].
  • variable length constructs were designed from the N-terminal region of l-Hmul that encompass the entire catalytic domain. Fragments were fused to the N-terminus of either l-Crel_NFSl or l-Crel_NFS2 to generate 8 constructs (SEQ ID NO: 9 and SEQ ID NO: 105-111).
  • each "enhanced" meganuclease was assessed using yeast assay described in International PCT Applications WO 2004/067736.
  • yeast assay described in International PCT Applications WO 2004/067736.
  • all constructs were screened against libraries of the C1221 target DNA ( Figure 2) having regions mutated in the following positions (numbering as in Figure 2): 12NN, lONNNnn, 7NNNnn, 5NNN, 2NN.
  • These targets contain nucleotide substitutions at the +/- positions indicated.
  • fusion proteins strongly enhanced cleavage of targets relative to the
  • Table 3 Activity in Yeast assay for l-Hmul/l-Crel fusions. The activity of the hHmuCre fusion protein relative to that of wild-type l-Crel is shown for various target libraries (see Example 3 text for details). Activity enhancement is based on a direct comparison of the change in activity for targets cleavable by the hHmuCre hybrid. The total number of targets in a given library is indicated next to the library name. The percent of targets enhanced to a given threshold are listed in each column. Assays were performed at two temperatures, 30°C and 37°C. Numbers in parentheses indicate actual target number enhanced versus total targets cleaved at the given temperature.
  • Table 4 Additional 2NN targets cleaved by hHmuCre at 37°C. From the 136 possible 2NN targets, hHmuCre is able to cleave a total of 125 (see Table 3) at 37°C. Of these, 13 DNA sequences are not cleaved by wild-type l-Crel at this temperature. Listed are the 2NN sequences and relative activities
  • Table 5 Additional 2NN targets cleaved by hHmuCre at 30"C. From the 136 possible 2NN targets, hHmuCre is able to cleave a total of 110 (see Table 3) at 30°C. Of these, 47 DNA sequences are not cleaved by wild-type l-Crel at this temperature. Listed are the 2NN sequences and relative activities for hHmuCre vs wild-type l-Crel.DNA sequence is based on the C1221target with the central four base pairs replaced as indicated. Relative activity is scaled as: -, no activity detectable; +, ⁇ 25% activity; ++, 25% to ⁇ 50% activity; +++, 50% to ⁇ 75% activity; ++++, 75% to 100% activity.
  • constructs were generated by adding variable length regions of the CFP1 peptide (SEQ ID NO: 112) to the C-terminus of the protein.
  • l-Crel is a homodimer
  • each "enhanced" meganuclease was assessed using yeast assay described in International PCT Applications WO 2004/067736 (see Example 1).
  • yeast assay described in International PCT Applications WO 2004/067736 (see Example 1).
  • all constructs were screened against libraries of the C1221 target DNA ( Figure 2) having regions mutated in the following positions (numbering as in Figure 2): 12NN, lONNNnn, 7NNNnn, 5NNN, 2NN.
  • These targets contain nucleotide substitutions at the +/- positions indicated. Fusion proteins enhance cleavage of targets to various degrees relative to the activity of wild-type l-Crel.
  • Hybrid restriction enzymes zinc finger fusions to Fok I cleavage
  • TAL nucleases hybrid proteins composed of TAL effectors
  • TAL nucleases hybrid proteins composed of TAL effectors
  • nbtl410 [pii] 10.1038/nbtl410.” Nature Biotechnology 26(7): 808-16.
  • thermostable archaeal intron- encoded endonuclease l-Dmol J Mol Biol 286(4): 1123-36.

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Abstract

La présente invention concerne un procédé d'augmentation de l'efficacité d'endonucléases à coupure rare, telles que des méganucléases. Plus particulièrement, la présente invention concerne un procédé de création de protéines de fusion qui consistent en un ou plusieurs domaines « de stimulation » fusionnés au moins à un échafaudage issu d'une endonucléase à coupure rare dans une chaîne polypeptidique unique pour une vectorisation simple et efficace. La présente invention concerne également des endonucléases à coupure rare, chimériques, génétiquement modifiées, des vecteurs, des compositions et des trousses utilisés pour mettre en œuvre le procédé et l'utilisation desdites endonucléases à coupure rare, chimériques, selon l'invention pour diverses applications allant du ciblage de gène homologue à la mutagenèse ciblée et à l'élimination de séquence.
PCT/US2012/032386 2011-04-05 2012-04-05 Procédé pour l'amélioration de l'efficacité d'endonucléases à coupure rare et ses utilisations Ceased WO2012138901A1 (fr)

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