WO2012138901A1 - Method for enhancing rare-cutting endonuclease efficiency and uses thereof - Google Patents

Abstract

The present invention relates to a method for enhancing the efficiency of rare-cutting endonucieases 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 endonucieases, vectors, compositions and kits used to implement the method and use of said chimeric rare-cutting endonucieases according to the invention for various applications ranging from homologous gene targeting to targeted mutagenesis and sequence removal.

Description

METHOD FOR ENHANCING RARE-CUTTING ENDONUCLEASE EFFICIENCY AND USES THEREOF

The present application claims priority to U.S. Serial number 61/472,053 filed April 5, 2011, the entire contents of which are incorporated herein by reference.

Field of the invention

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.

Background of the invention

Mammalian genomes constantly suffer from various types of damage, of which double-strand breaks (DSBs) are considered the most dangerous (Haber 2000). Repair of DSBs can occur through diverse mechanisms that can depend on cellular context. Repair via homologous recombination (HR) is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G2 phases of the cell cycle where the sister chromatids are in close proximity (Sonoda, Hochegger et al. 2006). Single-strand annealing (SSA) is another homology-dependent process that can repair DSBs between direct repeats and thereby promotes deletions (Paques and Haber 1999). Finally, non-homologous end joining (NHEJ) of DIMA is a major pathway for the repair of DSBs that can function throughout the cell cycle and does not depend on homologous recombination (Moore and Haber 1996; Haber 2008). NHEJ seems to comprise at least two different components: (i) a pathway that consists mostly in the direct re-joining of DSB ends, and which depends on the XRCC4, Lig4 and Ku proteins, and; (ii) an alternative NHEJ pathway, which does not depend on XRCC4, Lig4 and Ku, and is especially error- prone, resulting mostly in deletions, with the junctions occurring between micro-homologies (Frank, Sekiguchi et al. 1998; Gao, Sun et al. 1998; Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et al. 2007; Haber 2008; McVey and Lee 2008). Homologous gene targeting (HGT), first described over 25 years ago (Hinnen, Hicks et al. 1978; 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. To address these issues, an extensive assortment of rational approaches has been proposed with the intent of achieving greater than 1% targeted modifications. Many groups have focused on enhancing the efficacy of HGT, with two major disciplines having become apparent: (i) so-called "matrix optimization" methods, essentially consisting of modifying the targeting vector structure to achieve maximal efficacy, and; (ii) methods involving additional effectors to stimulate HR, generally sequence-specific endonucleases. The field of matrix optimization has covered a wide range of techniques, with varying degrees of success (Russell and Hirata 1998; Inoue, Dong et al. 2001; Hirata, Chamberlain et al. 2002; Taubes 2002; Gruenert, Bruscia et al. 2003; Sangiuolo, Scaldaferri et al. 2008; Bedayat, Abdolmohamadi et al. 2010). Stimulation of HR via nucleases, on the other hand, has repeatedly proven efficient (Paques and Duchateau 2007; Carroll 2008).

For 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. Alternatively, 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. 2009; Yang, Djukanovic et al. 2009). The nature of this process, which does not rely on site-specific or homologous recombination, gives rise to a third targeted approach based on endonuclease-induced mutagenesis. This approach, as well as the related applications, may be simpler than those based on homologous recombination in that (a) one does not need to introduce a repair matrix, and; (b) efficacy will be less cell-type dependant (in contrast to HR, NHEJ is probably active throughout the cell cycle (Delacote and Lopez 2008). 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). In addition, 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). Over the last 15 years, the use of meganucleases to successfully induce gene targeting has been well documented, starting from straightforward experiments involving wild-type l-Scel to more refined work involving completely re-engineered enzymes (Stoddard, Scharenberg et al. 2007; Galetto, Duchateau et al. 2009; Marcaida, Munoz et al. 2010; Arnould, Delenda et al. 2011). 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 αββαββα 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 α/β domains assemble to form the functional protein, with the β-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. 2006; Doyon, Pattanayak et al. 2006; Rosen, Morrison et al. 2006; Smith, Grizot et al. 2006; Arnould, Perez et al. 2007). In addition, whereas all known LAGLIDADG proteins analyzed to date act as "cleavases" to cut both strands of the target DNA, recent progress has been made in generating "mega-nickases" that cleave only one strand (Niu, Tenney et al. 2008; McConnell Smith, Takeuchi et al. 2009). Such enzymes can in principle provide similar levels of targeted induced HR with a minimization in the frequency of NHEJ. Although numerous engineering efforts have focused on 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). Analysis of the l-Basl HE revealed that although the N-terminal catalytic domain belongs to the HNH family, the C-terminal DNA-binding region resembles the intron-encoded endonuclease repeat motif (IENR1) found in endonucleases of the GIY-YIG family (Landthaler and Snub 2003). 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. 2004; Shen, Landthaler et al. 2004; Landthaler, Shen et al. 2006). Whereas the above families of proteins contain sequence-specific nucleases, the 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). As well as having the HNH motif, several of these nucleases contain the signature DRGH motif and share structural homology with core elements forming the ββα-Me-finger active site motif. Mutational studies of residues in the HNH/DRGH motifs have confirmed their role in nucleic acid cleavage activity (Ku, Liu et al. 2002; Doudeva, Huang et al. 2006; Eastberg, Eklund et al. 2007; Huang and Yuan 2007). Furthermore, the DNA binding affinity and sequence preference for ColE7 could be effectively altered (Wang, Wright et al. 2009). Such detailed studies illustrate the potential in re-engineering nonspecific nucleases for targeted purposes.

Zinc-finger nucleases (ZFNs), 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). In theory, one of the major advantages of ZFNs is that they 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). However, 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.

Recently, a new class of chimeric nuclease using a Fokl catalytic domain has been described (Christian, Cermak et al. 2010; Li, Huang et al. 2011). The DNA binding domain of these nucleases is derived from Transcription Activator Like Effectors (TALE), a family of proteins used in the infection process by plant pathogens of the Xanthomonas genus. In these DNA binding domains, sequence specificity is driven by a series of 33-35 amino acids repeats, differing essentially by two positions (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). 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). The apparent modularity of these DNA binding domains has been confirmed to a certain extent by modular assembly of designed TALE-derived protein with new specificities (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). However, one cannot yet rule out a certain level of context dependence of individual repeat/base recognition patterns, as was observed for Zinc Finger proteins (see above). Furthermore, it has been shown that natural TAL effectors can dimerize (Gurlebeck, Szurek et al. 2005) and how this would affect a "dimerization-based" TALE-derived nuclease is currently unknown.

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 (Christian, Cermak et al. 2010; Li, Huang et al. 2011). As such, 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).

In genome engineering experiments, 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).

Cleavage by peptidic rare cutting endonucieases usually generates cohesive ends, with 3' overhangs for LAGLIDADG meganucleases(Chevalier and Stoddard 2001) and 5' overhangs for Zinc Finger U 2012/032386

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). In mammalian cells, several experiment have shown that perfect re-ligation of compatible cohesive ends resulting from two independent but close l-Scel-induced DSBs is an efficient process (Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, ass et al. 2007; Bennardo, Cheng et al. 2008; Bennardo, Gunn et al. 2009). Absence of the Ku DNA repair protein does not significantly affect the overall frequency of NHEJ events rejoining the ends from the two DSBs; however it very strongly enhances the contribution of imprecise NHEJ to the repair process in CHO immortalized cells and mouse ES cells (Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et al. 2007; Bennardo, Cheng et al. 2008). Furthermore, the absence of Ku stimulates l-Scel-induced events such as imprecise NHEJ (Bennardo, Cheng et al. 2008), single-strand annealing (Bennardo, Cheng et al. 2008) and gene conversion (Pierce, Hu et al. 2001; Bennardo, Cheng et al. 2008) in mouse ES cells . Similar observations shave been made with cells deficient for the XRCC4 repair protein (Pierce, Hu et al. 2001; Guirouilh-Barbat, Rass et al. 2007; Bennardo, Gunn et al. 2009) (although XRCC4 deficiency affects the overal level of NHEJ in CHO cells (Guirouilh-Barbat, Rass et al. 2007)) or for DNA-PK (Pierce, Hu et al. 2001). In contrast, knock-down of CtIP has been shown to suppresses "alt-NHEJ" (a Ku- and XRCC4-independent form of NHEJ more prone to result in imprecise NHEJ), single-strand annealing and gene conversion, while not affecting the overall level of rejoining of two compatible ends generated by l-Scel (Bennardo, Cheng et al. 2008). Thus, competition between different DSB repair pathways can affect the spectrum or repair events resulting from a nuclease-induced DSB.

In addition, 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)). In eukaryotic cells 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). In addition, processing ends involving a damaged nucleotide (resulting from chemical cleavage or from a bulk adduct), requires the CtlP/Sae2 protein together with MN (Sartori, Lukas et al. 2007; Buis, Wu et al. 2008; Hartsuiker, Mizuno et al. 2009). Over-expression of the Trex2 exonuclease was shown to strongly stimulate imperfect NHEJ associated with loss of only a few base pairs (Bennardo, Gunn et al. 2009), while it inhibited various kinds of DNA repair events between distant sequences (such as Single-strand annealing, NHEJ between ends from different breaks, or NHEJ repair of a single DSB involving remote micro-homologies). In the same study, it was suggested that Trex2 did resect the 3' overhangs let by l-Scel in a non processive way. Thus, the type of stimulated pathway could in turn depend on the type of resection (length of resection, single strand vs. double strand, resection of 5' strand vs. 3' strand).

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). Furthermore, 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.

T/US2012/032386

Brief summary of the invention

In a general aspect, 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. In another aspect, 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. In another aspect, 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.

Brief description of the figures

In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, as well as to the appended drawings. A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below. 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.

Figure 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. As each end of the meganuclease scaffold is amenable to fusion, 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.

Figure 4 : Schematic of DNA cleavage, in vivo re-ligation and other repair pathways. In cells, cleavage by peptidic rare-cutting endonucleases usually result in a DNA double strand break (DSB) with cohesive ends. For example, 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. Thus, 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. Addition of two nickase activities affecting opposite strands (D) or of a new cleavase activity generating a second DSB (E) would result in a double strand gap. As a consequence, perfect re-ligation is not possible anymore, and one or several alternative repair outcomes could be stimulated. The current figure makes no assumption regarding the relative frequencies of these alternative outcomes (imprecise NHEJ, homologous recombination, others...). Solid triangles represent hydrolysis of phosphodiester bonds.

Detailed description of the invention

Unless specifically defined herein below, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology. 2386

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in- chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In a general aspect, 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. According to 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:

(i) Identifying at said genomic locus of interest one DNA target sequence cleavable by one rare-cutting endonuclease;

(ii) Engineering said rare-cutting endonuclease in order to create a chimeric rare-cutting endonuclease wherein said chimeric rare-cutting endonuclease is a fusion protein comprising at least a rare-cutting endonuclease-derived scaffold and at least one enhancer domain and wherein said chimeric rare-cutting endonuclease has an enhanced efficiency for said DNA target sequence relative to the efficiency of the starting rare-cutting endonuclease for said DNA target sequence;

(iii) Contacting said DNA target sequence with said chimeric rare-cutting endonuclease for cleavage within the genomic locus of interest; thereby obtaining a cell in which rare-cutting endonuclease efficiency at a genomic locus of interest in a cell is enhanced.

In an embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

In another preferred embodiment, said enhancer domain is catalytically active, or not providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold. In another preferred embodiment, said enhancer domain consists of a protein domain derived from an exonuclease. In a more preferred embodiment, 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.

In a more preferred embodiment, 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 (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66),, a functional mutant, a variant or a derivative of these protein domains thereof. In another more preferred embodiment, said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112). In another more preferred embodiment, 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), Endonuclease yncB, Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66), a functional mutant, a variant or a derivative of these protein domains thereof, can be fused to both N-terminus part of said rare- cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease- derived scaffold, respectively. For example, 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. In another more preferred embodiment, 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. In another preferred embodiment, 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.

In a preferred embodiment, said rare-cutting endonuclease-derived scaffold is derived from a meganuclease. In another preferred embodiment, said meganuclease comprises two identical monomers. In another preferred embodiment, said meganuclease comprises two non-identical monomers. In another preferred embodiment, said meganuclease is a single-chain meganuclease. In a more preferred embodiment, 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. In a more preferred embodiment, rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease- derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1). In another more preferred embodiment, 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. In another more preferred embodiment, 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. As a non-limiting example, 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. As a non-limiting example, 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. In another more preferred embodiment, 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). GENBANK/ NAME SEQ FASTA SEQUENCE

SWISS- ID

PROT ID NO

ACC85607.1 Mmel 10 >gi I 186469979 I gb| ACC85607.11 Mmel [Methylophilus methylotrophus]

NLALSWNEIRRKAIEFSKRWEDASDENSQAKPFLIDFFEVFGITNKRVATFEHAVKKFAKAHKEQSRGFVD

LFWPGILLIEMKSRGKDLDKAYDQALDYFSGIAERDLPRYVLVCDFQRFRLTDLITKESVEFLLKDLYQN

VRSFGFIAGYQTQVIKPQDPINIKAAERMGKLHDTLKLVGYEGHALELYLVRLLFCLFAEDTTIFEKSLF

QEYIETKTLEDGSD1_LAHHINTLFYVLNTPEQKRLKNLDEHIJ^FPYINGKLFEEPLPPAQFDKAMREALL

DLCSLDWSRISPAIFGSLFQSIMDAKKRRNLGAHYTSEANILKLIKPLFLDELWVEFEKVKNN NKLLAF

HKKLRGLTFFDPACGCGNFLVITYRELRLLEIEVLRGLHRGGQQVLDIEHLIQINVDQFFGIEIEEFPAQ

IAQVALWLTDHQMNMKISDEFGNYFARIPLKSTPHILNANALQIDWNDVLEAKKCCFILGNPPFVGKSKQ

TPGQKADLLSVFGNLKSASDLDLVAA YPKAAHYIQTNANIRCAFVSTNSITQGEQVSLLWPLLLSLGIK

INFAHRTFSWTNEASGVAAVHCVI IGFGLKDSDEKI IYEYESINGEPLAIKAKNINPYLRDGVDVIACKR

QQPISKLPSMRYGNKPTDDGNFLFTDEEK QFITNEPSSEKYFRRFVGGDEFINNTSRWCLWLDGADISE

IRAMPLVLARIKKVQEFRLKSSAKPTRQSASTPMKFFYISQPDTDYLLIPETSSENRQFIPIGFVDR VI

SSNATYHIPSAEPLIFGLLSSTMHNCWMR VGGRLESRYRYSASLVYNTFP IQPNEKQSKAIEEAAFAI

LKARSNYPNESLAGLYDPKTMPSELLKAHQKLDKAVDSVYGFKGPNTEIARIAFLFETYQKMTSLLPPEK

EIKKSKGK

Q47112.2 Colicin-E7 11 >gi|l2644448|sp|Q47112.2|CEA7_ECOLX RecName : Full=Colicin-E7

(CEA7_EC MSGGDGRGHNSGAHNTGGNINGGPTGLGGNGGASDGSG SSEN PWGGGSGSGVHWGGGSGHGNGGGNSN

OLX) SGGGSNSSVAAPMAFGFPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSA GIALYGILPSEIA

KDDPlnVIMSKIVTSLPAETVTl QVSTLPLDQATVSVTKRVTDVVKDTRQHIAVVAGVPMSVPVV AKPTR

TPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYV

SVTDVLTPAQVKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAELNQA KDVARNQERQAKAVQVYNSRK

SELDAA KTLADAKAEIKQFERFAREPMAAGHRMWQMAGLKAQRAQTDV KKAAFDAAAKEKSDADVA

SSALERRKQKENKEKDAKAKLDKESKRNKPGKATGKGKPV KWLN AGKDLGSPVPDRIANKLRDKEFK

SFDDFRKKFWEEVSKDPELSKQFSRInSINDRMKVGKAPKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDN

ISWTPKRHIDIHRGK

21/1

21/2

IADYIIAKLQHRDTSNIEQLLINKNLKMVEFLSKNTKNDNNFTYSE ESIYNGTYRITNLPSLGRFKFRK

KIAEKSLSGKVKEFNNIVQRYSVGLASSDLPFGVIRKESRNDFINDVCKLYNINDMKI IKELKEDADLIV CMLKGFKPRGDDNRPDRGALPLVA LAGENAQIFTFIYGPLIKGAINLIDQDINKLAKRNGLWKSFVSLS DFIVLDCPI IGESYNEFRLI INKNNKESILRKTSKQQNILVDPTPNHYQENDVDTVIYSIFKYIVPNCFS GMCNPPGGDWSGLSI IRNGHEFRWLSLPRVSENGKRPDHVIQILDLFEKPLLLSIESKEKPNDLEPKIGV QLIKYIEYLFDFTPSVQRKIAGGN EFGNKSLVPNDFILLSAGAFIDYDNLTENDYEKIFEVTGCDLLIA IKNQNNPQKWVIKFKPKNTIAEKLVNYIKLNFKSNIFDTGFFHIEG

ADI24225.1 Nb.BtsCI 46 >gi I 297185870 |gb| ADI24225.11 BtsCI bottom- strand nicking enzyme variant

[synthetic construct]

MKRILYLLTEERPKINIIHQI INLEYKATLHFGAKIVPVMNEENKFTFIYHVKGIEVEGFDAVLIKIVSG HSSFVDYLVFDSNDLKPEKNTITLFDLDQYELDLSYYFGKGWIVRIPSPSDLPKYWEETKTDDHESRNT NAYQRSSKFVFCELYYGKEVKKYMLYDISDGRTLSGTDTHNFGMR LVTNNVNLVGVPN YLPFTDIKEF INEKNRIADNGPSHNVPIRLKLDKEKNVIYISAKLDKGNGKNKNKISNDPNIGAVAI ISATLRNLNWKGD IEIINHNLLPSSISSRSNGNKLLYIMKKLGVRFNNII NWNNIKNNINYFFYNITSEKIVSIYYHLYVED KLSNARVIFDNHAGCGKSYFRTLNNKI IPVGKEIPLPALVIFDSDQNIVKVIAAAKAENVYNGVEQLSTF DKFIESYINKYYPGAAVECSVITWGKSSNPYVSFYLDKDGSAVFL

ADI24224.1 N BtsCI 47 >g I 297185868 |gb| ADI24224.11 BtsCI top-strand nicking enzyme variant

[synthetic construct]

MKRILYLLTEERPKINIIHQIINLEYKATLHFGAKIVPVMNEENKFTFIYHVKGIEVEGFDAVLIKIVSG HSSFVDYLVFDSNDLKPEKNTITLFDLDQYELDLSYYFGKGWIVRIPSPSDLPKYWFETKTDDHESRNT NAYQRSSKFVFCELYYGKEVKKYMLYDISDGRTLSGTDTHNFGMRMLVTNNVNLVGVPNMYLPFTDIKEF INEKNRIADNGPSHNVPIRLKLDKEKNVIYISAKLDKGNGKNKNKISNDPNIGAVAIISATLRNLNWKGD IEIINHNLLPSSISSRSNGNKLLYIMKKLGVRFNNINVNWNNIKNNINYFFYNITSEKIVSIYYHLYVED KLSNARVIFDNHAGCGKSYFRTLNNKI IPVGKEIPLPDLVIFDSDQNIVKVIEAEKAENVYNGVEQLSTF DKFIESYINKYYPGAAVECSVITWGKSSNPYVSFYLDKDGSAVFL

>gi I 85720924 IgbIABC75874.11 Rl.BtsI [Geobacillus thermoglucosidasius] MKITEGIVHVAMRHFLKSNGWKLIAGQYPGGSDDELTALNIVDPWARDNSPDPRRHSLGKIVPDLIAYK NDDLLVIEAKPKYSQDDRDKLLYLLSERKHDFYAALEKFATERNHPELLPVSKLNIIPGLAFSASENKFK KDPGFVYIRVSGIFEAFMEGYDWG

ABC75874.1 Rl.BtsI 48 >gi I 85720924 IgbIABC75874.11 Rl.BtsI [Geobacillus thermoglucosidasius]

21/3

21/4

21/5

RSAELSEDDLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRK

NEESGAWVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSS DDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTAL QQFRRKSDSPTSLPE MSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDS DSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKP ASIQKRKHHNAENKPGLQIKLNEL KNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRV QRAIFQ

P39875.2 Yeast Exol 57 1706421 | | P39875.2 | EX01_YEAST RecName : Full=Exodeoxyribonuclease

(EX01_YE 1; AltName: Full=Exodeoxyribonuclease I; Short=EXO I; Short=Exonuclease AST) I; AltName: F ll=Protein DHS1

MGIQGLLPQLKPIQNPVSLRRYEGEVLAIDGYAWLHRAACSCAYELAMGKPTDKYLQFFIKRFSLLKTFK VEPYLVFDGDAIPVKKSTESK-RRDKRKENKAIAERLWACGEKKNAMDYFQKCVDITPEMAKCI ICYCKLN GIRYIVAPFEADSQMVYLEQKNIVQGIISEDSDLLVFGCRRLITKLNDYGECLEICRDNFIKLPKKFPLG SLTNEEIITMVCLSGCDYTNGIPKVGLITA KLVRRFNTIERIILSIQREGKLMIPDTYINEYEAAVLAF QFQRVFCPIRKKIVSLNEIPLYLKDTESKRKRLYACIGFVIHRETQKKQIVHFDDDIDHHLHLKIAQGDL NPYDFHQPLANREHKLQILASKSNIEFGKTNTTNSEAKVKPIESFFQKMTKLDHNPKVANNIHSLRQAEDK LT1XLAIKRRKLSNANVVQETLKDTRSKFFNKPSMTVVENFKEKGDSIQDFKEDTNSQSLEEPVSESQLSTQ IPSSFITTNLEDDDNLSEEVSEWSDIEEDRKNSEGKTIGNEIYNTDDDGDGDTSEDYSETAESRVPTSS TTSFPGSSQRSISGCTKVLQKFRYSSSFSGVNANRQPLFPRHVNQKSRGMVYVNQNRDDDCDDNDGKNQI TQRPSLRKSLIGARSQRIVIDMKSVDERKSFNSSPILHEESKKRDIETTKSSQARPAVRSISLLSQFVYK GK

BAJ43803.1 E.coli Exol 58 >gi 315136644 dbj BAJ43803.1 exonuclease I [Escherichia coli DH1]

MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEF VIGEPEVFYCKPADDYLPQPGAVLITGIT PQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDV MRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFT HRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTS VAPLA HPENR AVIMVDLAGDISPLLELDSDTLR ERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKWAI FAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLD YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIV

Q9BQ50.1 Human 59 >gi 47606206 s Q9BQ50.1 TREX2_HUMAN RecName: Full=Three prime repair

21/6

21/7

MGSQALPHGHMQTLIFLDLEATGLPYSQPKITELCLLAVHRHALENSSMSEGQPPPVPKPPRWDKLSLC

IAPGKPCSSGASEITGLTTAGLEAHGRQRFNDNLATLLQVFLQRQPQPCCLVAHNGDRYDFPLLQAELAS LSVISPLDGTFCVDSIAALKTLEQASSPSEHGPRKSYSLGSIYTRLYGQAPTDSHTAEGDVLALLSICQW KPQALLQWVDKHARPFSTIKPMYGMAATTGTASPRLCAATTSSPLATANLSPSNGRSRGKRPTSPPPENV PEAPSREGLLAPLGLLTFLTLAIAVLYGIFLASPGQ

AAH63664.1 Human 64 >gi I39793966 |gb|AAH63664.1 I DNA2 protein [Homo sapiens]

DNA2 FAIPASRMEQLNELELLMEKSFWEEAELPAELFQKKWASFPRTVLSTGMDNRYLVLAWTVQNKEGNCE

KRLVITASQSLENKELCILRNDWCSVPVEPGD11HLEGDCTSDT 11DKDFGYLILYPDMLISGTSIASS IRCMRRAVLSETFRSSDPATRQ LIGTVLHEVFQKAINNSFAPEKLQELAFQTIQEIRHLKEMYRLNLSQ DEIKQEVEDYLPSFCK AGDFMHKNTSTDFPQMQLSLPSDNSKDNSTCNIEWKP DIEESIWSPRFGLK GKIDVTVGVKIHRGYKTKYKIMPLELKTGKESNSIEHRSQWLYTLLSQERRADPEAGLLLYLKTGQMYP VPANHLDKRELLKLR QMAFSLFHRISKSATRQKTQLASLPQIIEEEKTCKYCSQIGNCALYSRAVEQQM DCSSVPIVMLPKIEEETQHLKQTHLEYFSLWCLMLTLESQSKDNKK HQNIWLMPASEMEKSGSCIGNLI RMEHVKIVCDGQYLHNFQCKHGAIPVTNLMAGDRVIVSGEERSLFALSRGYVKEIN TTVTCLLDRNLSV LPESTLFRLDQEEKNCDIDTPLGNLSKLMENTFVSKKLRDLI IDFREPQFISYLSSVLPHDAKDTVACIL KGLNKPQRQAMKKVLLSKDYTLIVGMPGTGKTTTICTLVPAPEQVEKGGVS VTEAKLIVFLTSIFVKAG CSPSDIGI IAPYRQQLKI INDLLARSIGMVEVNTVDKYQGRDKSIVLVSFVRSNKDGTVGELLKDWRRLN VAITRAKHKLILLGCVPSLNCYPPLEKLLNHLNSEKLI IDLPSREHESLCHILGDFQRE

P38859.1 Yeast 65 >giI 731738 I spIP38859.1 |DNA2_YEAST RecName : Full=DNA replication ATP- DNA2 dependent helicase DNA2

(DNA2 YE MPGTPQKNKRSASISVSPAKKTEEKEIIQNDSKAILSKQTKRKKKYAFAPINNLNGKNTKVSNASVLKSI AST) AVSQVRNTSRTKDINKAVSKSVKQLPNSQVKPKREMSNLSRHHDFTQDEDGPMEEVIWKYSPLQRDMSDK

TTSAAEYSDDYEDVQNPSSTPIVPNRLKTVLSFTNIQVPNADVNQLIQENGNEQVRPKPAEISTRESLRN IDDILDDIEGDLTIKPTITKFSDLPSSPIKAPNVEKKAEVNAEEVDKMDSTGDSNDGDDSLIDILTQKYV EKRKSESQITIQGNTNQKSGAQESCGKNDNTKSRGEIEDHENVDNQAKTGNAFYENEEDSNCQRIKKNEK IEYNSSDEFSDDSLIELLNETQTQVEPNTIEQDLDKVEKMVSDDLRIATDSTLSAYALRAKSGAPRDGW RLVIVSLRSVELPKIGTQKILECIDGKGEQSSVVVRHPWVYLEFEVGDVIHI IEGKNIENKRLLSDDKNP KTQLANDNLLVLNPDVLFSATSVGSSVGCLRRSILQMQFQDPRGEPSLVMTLGNIVHELLQDSIKYKLSH NKISMEI I IQKLDSLLETYSFSII ICNEEIQYVKELVMKEHAENILYFVNKFVSKSNYGCYTSISGTRRT QPISISNVIDIEENIWSPIYGLKGFLDATVEANVENNKKHIVPLEVKTGKSRSVSYEVQGLIYTLLLNDR YEIPIEFFLLYFTRDKNMTKFPSVLHSIKHILMSRNRMSMNFKHQLQEVFGQAQSRFELPPLLRDSSCDS

21/8

In a preferred embodiment, 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. In a more preferred embodiment, said peptidic linker sequence is selected from the group consisting of

NFSl, NFS2, CFSl, M2, BQY, QGPSG, LGPDG KA, la8h_l, ldnpA_l, ld8cA_2, lckqA_3, lsbp_l, lev7A_l, lalo_3, lamf l, ladjA_3, lfcdC_l, lal3_2, lg3p_l, lacc_3, lahjB_l, lacc_l, laf7_l, lheiA_l, lbia_2, ligtB_l, lnfkA_l, lau7A_l, lbpoB_l, lb0pA_2, lc05A_2, lgcb_l, lbt3A_l, lb3oB_2, 16vpA_6, ldhx_l, lb8aA_land lqu6A_l, as listed in Table 2 (SEQ ID NO: 67 to SEQ ID NO: 104). In a more preferred embodiment, 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). In the scope of the present invention is also encompassed the case where 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.

21/10 lbia_2 268 - 276 9 16,089 LDNFINRPV 84 l igtB l 1 1 1 - 1 19 9 19,737 VSSAKTTAP 85 lnfkA l 239 - 248 10 13,228 DSKAPNASNL 86 lau7A 1 103 - 1 12 10 20,486 KRRTTISIAA 87 lbpoB l 138 - 148 1 1 21,645 PVKMFDRHSSL 88 lb0pA_2 625 - 635 1 1 26,462 APAETKAEPMT 89 lc05A 2 135 - 148 14 23,819 YTRLPERSELPAEI 90 lgcbj 57 - 70 14 27,39 VSTDSTPVTNQ SS 91 lbt3A_l 38 - 51 14 28,818 YKLPAVTTM VRPA 92 lb3oB_2 222 - 236 15 20,054 IARTDLKKNRDYPLA 93

16vpA 6 312 - 332 21 23,713 TEEPGAPLTTPPTLHGNQARA 94 l dhx l 81 - 101 21 42,703 ARFTLAVGDNRVLDMASTYFD 95 lb8aA_l 95 - 120 26 31 ,305 IVVLNRAETPLPLDPTG VKAELDTR 96 lqu6A 1 79 - 106 28 51 ,301 ILNKE AVSPLLLTTTNSSEGLSMGNY 97

NFS1 - 20 - GSDITKSKISEKMKGQGPSG 98

NFS2 - 23 - GSDIT SKISEKMKG LGPDGRKA 99

CFS1 - 10 - SLTKSKISGS 100

RM2 - 32 - AAGGSALTAGALSLTAGALSLTAGALSGGGGS 101

BQY - 27 - AAGASSVSASGH1APLSLPSSPPSVGS 102

QGPSG - 5 - QGPSG 103

LGPDGR A - 8 - LGPDGRKA 104

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. In a preferred embodiment, said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease. In a more preferred embodiment, 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.

22 32386

Cleavage by rare-cutting endonucleases usually generates cohesive ends, with 3' overhangs for LAGLIDADG meganucleases (Chevalier and Stoddard 2001) and 5' overhangs for Zinc Finger Nucleases (Smith, Bibikova et al. 2000) These ends, which result from hydrolysis of phosphodiester bonds, can be re-ligated in vivo by N HEJ 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). In a preferred embodiment, enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, 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). 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. Thus, 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. As a non-limiting example, 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. As a consequence, 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. As another non-limiting example, 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.

In this first aspect of the invention, 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. In a preferred embodiment, 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. In another preferred embodiment, said structural support by at least one enhancer domain enhances the existing catalytical activity of a chimeric rare-cutting

23 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. In another preferred embodiment, 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. Figure 4

Depending on number (one to four) of enhancer domains to the starting rare-cutting endonuclease-derived scaffold according to the invention and on their catalytic activities (functional support) or not (structural support), the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention. As a non-limiting example, 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). As another non-limiting example, 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). As a non-limiting example, 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). As another non-limiting example, 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.

In this aspect of the present invention, 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

24 12 032386 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. Importantly, enhancement of efficacy in cells (enhanced generation of targeted mutagenesis or targeted recombination) can be, but is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays. For example, 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. However, as explained above, and in the legend of Figure 4, 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%.

In another preferred embodiment, 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. By 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. For instance, 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. In another preferred embodiment, 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. As a non limiting

25 example, 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.

In 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).

According to the present invention, efficiency of rare-cutting endonucleases such as meganucleases can be enhanced through the addition of a domain to promote existing or alternate activities. As each end of the meganuclease scaffold, for instance, is amenable to fusion, 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.

In a preferred embodiment, 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. In another more preferred embodiment, 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. In another more preferred embodiment, 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. In another more preferred embodiment, the addition of at least

26 two cataiyticaily active cleavase enhancer domains according to the invention 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.

In another preferred embodiment, 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. In a more preferred embodiment, 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. In another more preferred embodiment, 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.

In another preferred embodiment, 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.

In a more preferred embodiment, 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.

27 In another aspect, 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.

According to this aspect of the invention is a method to create a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence comprising the steps of:

(i) Engineering a rare-cutting endonuclease-derived scaffold;

(ii) Determining or engineering an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease when fused to it;

(iii) Optionally determining or engineering a peptidic linker to fuse said rare-cutting endonuclease-derived scaffold to said enhancer domain; thereby obtaining a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence.

In an embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

In a preferred embodiment, said enhancer domain is catalytically active or not, providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold. In another preferred embodiment, said enhancer domain consists of a protein domain derived from an exonuclease. In a

28 more preferred embodiment, 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.

In a more preferred embodiment, 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 (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66), a functional mutant, a variant or a derivative of these protein domains thereof. In another more preferred embodiment, said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112). In another more preferred embodiment, 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), Endonuclease yncB, Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1), a functional mutant, a variant or a derivative of these protein domains thereof, can be fused to both N-terminus part of said rare-cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease-derived scaffold, respectively. For example, 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. In another more preferred

29 P T/US2012/032386 embodiment, 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. In another preferred embodiment, 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.

In a preferred embodiment, said rare-cutting endonuclease-derived scaffold is derived from a meganuclease. In another preferred embodiment, said meganuclease comprises two identical monomers. In another preferred embodiment, said meganuclease comprises two non-identical monomers. In another preferred embodiment, said meganuclease is a single-chain meganuclease. In a more preferred embodiment, 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. In a more preferred embodiment, rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease- derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1). In another more preferred embodiment, 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. In another more preferred embodiment, 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. As a non-limiting example, 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. As a non-limiting example, 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. In another more preferred embodiment, 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).

In a preferred embodiment, 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. In a more preferred embodiment, said peptidic linker sequence is selected from the group consisting of 2012/032386

NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDGRKA, la8h_l, ldnpA_l, ld8cA_2, lckqA_3, lsbp_l, lev7A_l, lalo_3, lamf_l, ladjA_3, lfcdC_l, lal3_2, lg3p_l, lacc_3, lahjB_l, lacc_l, laf7_l, lheiA_l, lbia_2, ligtB_l, lnfkA_l, lau7A_l, lbpoB_l, lb0pA_2, lc05A_2, lgcb_l, lbt3A_l, lb3oB_2, 16vpA_6, ldhx_l, lb8aA_land lqu6A_l, as listed in Table 2 (SEQ ID NO: 67 to SEQ ID NO: 104). In a more preferred embodiment, 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). In the scope of the present invention is also encompassed the case where 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.

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. In a preferred embodiment, said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease. In a more preferred embodiment, 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). In a preferred embodiment, enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, can be the hydrolysis of additional

31 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). 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. Thus, 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. As a non-limiting example, 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. As a consequence, 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. As another non-limiting example, 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.

In this second aspect of the invention, 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. In a preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, 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.

32 Depending on number (one to four) of enhancer domains to the starting rare-cutting endonuclease- derived scaffold according to the invention and on their catalytic activities (functional support) or not (structural support), the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention. As a non-limiting example, 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). As another non- limiting example, 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). As a non-limiting example, 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). As another non-limiting example, 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.

In this aspect of the present invention, 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. Importantly, enhancement

33 of efficacy in cells (enhanced generation of targeted mutagenesis or targeted recombination) is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays. For example, 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. However, as explained above, and in the legend of Figure 4, 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%. In another preferred embodiment, 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. By 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. For instance, 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. In another preferred embodiment, 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. As a non limiting example, 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.

34 2012/032386

In another aspect, 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.

According to this aspect of the invention is a chimeric rare-cutting endonuclease for a DNA target sequence comprising:

(i) a rare-cutting endonuclease-derived scaffold;

(ii) an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease for said DNA target sequence when fused to it;

(iii) Optionally a peptidic linker.

In an embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

In a preferred embodiment, said enhancer domain is catalytically active, providing functional and/or structural support to said rare-cutting endonuclease-derived scaffold. In a more preferred embodiment, said enhancer domain consists of a protein domain derived from an exonuclease. In another more preferred embodiment, said enhancer domain consists of a protein domain derived

35 from an endonuclease. In a 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. In a more preferred embodiment, 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 (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66), a functional mutant, a variant or a derivative of these protein domains thereof. In another more preferred embodiment, said enhancer domain consists of a peptide derived from CFPl peptide (SEQ ID NO: 112). In another more preferred embodiment, 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), Endonuclease yncB, Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6l (R.BspD6l large subunit), ss.BspD6l (R.BspD6l small subunit), R.PIel, Mlyl, Alwl, Mval269l, Bsrl, Bsml, Nb.BtsCI, Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1, BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 1 (SEQ ID NO: 10 to SEQ ID NO: 66), a functional mutant, a variant or a derivative of these protein domains thereof, can be fused to both N-terminus part of said rare- cutting endonuclease-derived scaffold and C-terminus part of said rare-cutting endonuclease- derived scaffold, respectively. For example, 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. In another more preferred embodiment, said enhancer domain consists of a catalytically active derivative of the protein domains listed above

36 and in Table 1, providing functional and/or structural support to said rare-cutting endonuclease- derived scaffold. In another preferred embodiment, 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.

In a preferred embodiment, said rare-cutting endonuclease-derived scaffold is derived from a meganuclease. In another preferred embodiment, said meganuclease comprises two identical monomers. In another preferred embodiment, said meganuclease comprises two non-identical monomers. In another preferred embodiment, said meganuclease is a single-chain meganuclease. In a more preferred embodiment, 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. In a more preferred embodiment, rare-cutting endonuclease-derived scaffold is a truncated form of wild- type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease- derived scaffold comprises first 152, 153, 154 or 155 amino acids residues of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 153 of wild-type l-Crel (SEQ ID NO: 1). In another more preferred embodiment, rare-cutting endonuclease-derived scaffold comprises residues 2 to 155 of wild-type I- Crel (SEQ ID NO: 1). In another more preferred embodiment, 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. In another more preferred embodiment, 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. As a non-limiting example, 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. As a non-limiting example, 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. In another more preferred embodiment, 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)..

In a preferred embodiment, 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. In a more preferred embodiment, said peptidic linker sequence is selected from

37 the group consisting of NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDG KA, la8h_l, ldnpA_l, ld8cA_2, lckqA_3, lsbp_l, lev7A_l, lalo_3, lamf_l, ladjA_3, lfcdC_l, lal3_2, lg3p_l, lacc_3, lahjB_l, lacc_l, laf7_l, lheiA_l, lbia_2, ligtB_l, lnfkA_l, lau7A_l, lbpoB_l, lb0pA_2, lc05A_2, lgcb_l, lbt3A_l, lb3oB_2, 16vpA_6, ldhx_l, lb8aA_land lqu6A_l, as listed in Table 2 (SEQ ID NO: 67 to SEQ ID NO: 104). In a more preferred embodiment, 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). In the scope of the present invention is also encompassed the case where 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.

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. In a preferred embodiment, said functional support can be the consequence of the hydrolysis of additional phosphodiester bonds. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from a nuclease. In a more preferred embodiment, said functional support can be the hydrolysis of additional phosphodiester bonds by a protein domain derived from an endonuclease. In a more preferred embodiment, 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). In a preferred embodiment, enhancement of efficiency of a chimeric rare-cutting endonuclease according to the present invention, compared to a starting rare-cutting endonuclease, can be the hydrolysis of additional

38 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). 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. Thus, 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. As a non-limiting example, 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. As a consequence, 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. As another non-limiting example, 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.

In this third aspect of the invention, 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. In a preferred embodiment, 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. In another preferred embodiment, 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. In another preferred embodiment, 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

39 enhanced efficiency for a DNA target at a genomic locus of interest, according to the present invention.

Depending on number (one to four) of enhancer domains to the starting rare-cutting endonuclease- derived scaffold according to the invention and on their catalytic activities (functional support) or not (structural support), the resulting chimeric rare-cutting endonuclease can comprise several enzymatic activities that contribute to the enhancement of its efficiency according to the present invention. As a non-limiting example, 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). As another non- limiting example, 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). As a non-limiting example, 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). As another non-limiting example, 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.

In this aspect of the present invention, 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

40 recombination can be measured by a cell-based recombination assay as described in the International PCT Application WO 2004/067736, as non-limiting example. Importantly, enhancement of efficacy in cells (enhanced generation of targeted mutagenesis or targeted recombination) can be, but is not necessarily associated with an enhancement of the cleavage activity that could be detected in certain in vitro assays. For example, 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. However, as explained above, and in the legend of Figure 4, 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%.

In another preferred embodiment, 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. By 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. For instance, 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. In another preferred embodiment, 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. As a non limiting example, 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.

In this third aspect of the invention 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

41 NO: 108), hHmuCre_D0202 (SEQ ID NO: 109), hHmuCre_D0302 (SEQ ID NO: 110) and hHmuCre_D0402 (SEQ ID NO: 111).

In another aspect, 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.

In another preferred embodiment, 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.

For purposes of therapy, 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. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality. 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").

In one further aspect of the present invention, 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. In a preferred embodiment, 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

42 Davis et al. (US 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble chimeric rare-cutting endonuclease conjugates with anti-viral activity. Similar methods also using a polyethylene-polypropylene glycol copolymer are described in Saifer ef al. (US 5,006,333). The present invention also relates to vectors, compositions and kits used to implement the method. More specifically, 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.

43 Definitions

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

- 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 according to the present invention 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. For chimeric rare-cutting endonucleases derived from LAGLIDADG meganuclease subfamily, 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. As non-limiting example, an

44 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. Also, 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.

- Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.

- by "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.

- by "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

45 which interacts with the other half of the DNA target to form a functional meganuclease able to cleave said DNA target.

- by "endonuclease variant", "rare-cutting endonuclease variant", chimeric rare-cutting endonuclease variant" or "meganuclease variant" or "variant" it is intended an endonuclease, rare- cutting endonuclease, chimeric rare-cutting endonuclease or meganuclease obtained by replacement of at least one residue in the amino acid sequence of the parent endonuclease, rare- cutting endonuclease, chimeric rare-cutting endonuclease or meganuclease, i.e. 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.

- 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.

Generally, 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.

- by "related to", particularly in the expression "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.

- by "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. Following cleavage of the DNA target, 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

46 12 032386 genomic sequence containing the DNA target is replaced by the third portion of the repair matrix and a variable part of the first and second portions of the repair matrix.

- by "functional variant" is intended a catalytically active variant of a protein, such variant can have additional properties compared to its parent protein. As a non-limiting example, a functional variant of a meganuclease can be able to cleave additional DNA target sequences that are not cleaved by the parent meganuclease.

- by "derived from" or "derivative(s)" it is intended to mean for instance 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. In this definition, 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.

- by "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. In the present Patent

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. These additional residues do not affect the properties of the enzyme and to avoid confusion these additional residues do not affect the numeration of the residues in l-Cre\ or a variant referred in the present Patent Application, as these references exclusively refer to residues of the wild type l-Cre\ enzyme (SEQ ID NO: 1) as present in the variant, so for instance residue 2 of /- Crel is in fact residue 3 of a variant which comprises an additional Alanine after the first Methionine.

- by "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₊₁c₊₂g₊3a₊ C₊5g₊₆t₊7t₊8t₊₉t₊i₀g_{+ 1}ia₊₁₂ (SEQ ID NO: 2), also called C1221.

- by "domain" or "core domain" is intended the "LAG LIDADG homing endonuclease core domain" which is the characteristic αββαββα fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues. Said domain comprises four beta-strands (β^β_,ιβ^ 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. For example, in the case of the dimeric homing

47 endonuclease l-Crel (163 amino acids), the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.

- by "subdomain" is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half-site.

- by "beta-hairpin" is intended two consecutive beta-strands of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease core domain (βιβ₂ 0Γ β₃ β₄ ) which are connected by a loop or a turn,

- by "single-chain meganuclease", "single-chain chimeric meganuclease", "single-chain meganuclease derivative", "single-chain chimeric meganuclease derivative" or "single-chain derivative" is intended a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic 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.

- by "DNA target", "DNA target sequence", "target DNA sequence", "target sequence" , "target-site", "target" , "site", "site of interest", "recognition site", "polynucleotide recognition site", "recognition sequence", "homing recognition site", "homing site", "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. These terms refer to 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. For the LAGLIDADG subfamily of rare-cutting endonucleases, 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

48 sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an l-Crel-derived variant occurs, corresponds to the cleavage site on the sense strand of the DNA target. - by "DNA target half-site", "half cleavage site" or half-site" is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain.

- The term "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. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). 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). In chemical endonucleases, 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). 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. As such, 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 TALENs.

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

49 12 032386 to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease.

In the wild, meganucleases are essentially represented by homing endonucleases. Homing Endonucleases (HEs) are a widespread family of natural meganucleases including hundreds of proteins families (Chevalier and Stoddard 2001). These proteins are encoded by mobile genetic elements which propagate by a process called "homing": the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus. Given their exceptional cleavage properties in terms of efficacy and specificity, they could represent ideal scaffolds to derive novel, highly specific endonucleases.

HEs belong to five major families. 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.

Although the 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. 1999) or \-Ani\ (Bolduc, Spiegel et al. 2003). Both monomers and both domains (for monomeric proteins) contribute to the catalytic core, organized around divalent cations. Just above the catalytic core, the two LAGLIDADG peptides also play an essential role in the dimerization interface. DNA binding depends on two typical saddle-shaped αββαββα folds, sitting on the DNA major groove. Other domains can be found, for example in inteins such as PI-P/ul (lchiyanagi, Ishino et al. 2000) and Pl-Scel (Moure, Gimble et al. 2002), whose protein splicing domain is also involved in DNA binding.

The making of functional chimeric meganucleases, by fusing the N-terminal l-Dmol domain with an l-Crel monomer (Chevalier, Kortemme et al. 2002; Epinat, Arnould et al. 2003); International PCT Application WO 03/078619 (Cellectis) and WO 2004/031346 (Fred Hutchinson Cancer Research Center, Stoddard et al)) have demonstrated the plasticity of LAGLIDADG proteins.

Different groups have also used a semi-rational approach to locally alter the specificity of the l-Crel (Seligman, Stephens et al. 1997; Sussman, Chadsey et al. 2004); International PCT Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO 2007/049156 (Cellectis); (Arnould,

50 12 032386

Chames et al. 2006; Rosen, Morrison et al. 2006; Smith, Grizot et al. 2006), l-5cel (Doyon, Pattanayak et al. 2006), Pl-Scel (Gimble, Moure et al. 2003) and l-Msol (Ashworth, Havranek et al. 2006).

In addition, hundreds of l-Crel derivatives with locally altered specificity were engineered by combining the semi-rational approach and High Throughput Screening:

- Residues Q44, R68 and R70 or Q44, R68, D75 and 177 of l-Crel were mutagenized and a collection of variants with altered specificity at positions ± 3 to 5 of the DNA target (5NNN DNA target) were identified by screening (International PCT Applications WO 2006/097784 and WO 2006/097853 (Cellectis); (Arnould, Chames et al. 2006; Smith, Grizot et al. 2006).

- Residues K28, N30 and Q38 or N30, Y33 and Q38 or K28, Y33, Q38 and S40 of l-Crel were mutagenized and a collection of variants with altered specificity at positions ± 8 to 10 of the DNA target (10NNN DNA target) were identified by screening (Arnould, Chames et al. 2006; Smith, Grizot et al. 2006); International PCT Applications WO 2007/060495 and WO 2007/049156 (Cellectis)).

Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of two different halves of each variant DNA target sequence ((Arnould, Chames et al. 2006; Smith, Grizot et al. 2006); International PCT Applications WO 2006/097854 and WO 2007/034262).

Furthermore, 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 mutations from the two subdomains of l-Crel within the same monomer allowed the design of novel chimeric molecules (homodimers) able to cleave a palindromic combined DNA target sequence comprising the nucleotides at positions ± 3 to 5 and ± 8 to 10 which are bound by each subdomain ((Smith, Grizot et al. 2006); International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)).

The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity are described in the International PCT Application WO 2004/067736; (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006). These assays result in a functional LacZ reporter gene which can be monitored by standard methods.

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. In a first step, couples of novel meganucleases are combined in new molecules

51 ("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. The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from different genes has been described in the following Cellectis International patent applications: XPC gene (WO2007/093918), RAG gene (WO2008/010093), HPRT gene (WO2008/059382), beta-2 microglobulin gene (WO2008/102274), Rosa26 gene (WO2008/152523), Human hemoglobin beta gene (WO2009/13622) and Human interleukin-2 receptor gamma chain gene (WO2009019614).

These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields, including gene therapy.

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-Ssp I, Pl-Fac I, Pl-Mja I, Pl-Pho I, Pi-Tag I, Pl-Thy I, Pl-Tko I, Pl-Tsp I, l-Msol.

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. Said substitution(s) can for example be introduced by site-directed mutagenesis and/or by random mutagenesis. In the frame of the present invention, such variant meganucleases, endonucleases or rare-cutting endonucleases remain functional, i.e. they retain the capacity of recognizing (binding

52 function) and optionally specifically cleaving a target sequence to initiate a gene engineering process. 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.

- by "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.

- By " 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. It includes, but is not limited to 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. These 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. By "delivery vector" or "delivery vectors" is also intended delivery methods to perform transfection.

- The terms "vector" or "vectors" refer 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. g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

- By "lentiviral vector" is meant 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. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, 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.

54 2012/032386

By "integrative lentiviral vectors (or LV)", is meant such vectors as non limiting example, that are able to integrate the genome of a target cell.

At the opposite by "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. In general, 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. Large numbers of suitable vectors are known to those of skill in the art. 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. Preferably 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. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-p-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), ct-antitrypsin protease, human surfactant (SP) A and B proteins, β- casein and acidic whey protein genes.

55 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.

By cell or cells is intended any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.

- By "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.

In the frame of the present invention, "eukaryotic cells" refer 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.

More preferably 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, Cucurbita pepo, Citrullus lanatus, Cucumis melo, Citrus aurantifolia, Citrus maxima, Citrus medica, Citrus reticulata.

More preferably 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

56 taurus, Danio rerio, Canis lupus, Felis catus, Equus caballus, Salmo salar, Oncorhynchus mykiss, Gallus gallus, Meleagris gallopavo, Drosophila melanogaster, Caenorhabditis elegans.

In the present invention, 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. As non limiting examples 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). 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.

- by "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.

- by "mutation" 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.

57 - In the frame of the present invention, the expressions "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

- By "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.

- As used herein, the term "transgene" refers to a sequence encoding a polypeptide.

Preferably, 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. Most preferably, the transgene encodes a therapeutic polypeptide useful for the treatment of an individual.

- The term "gene of interest" or "GOI" refers to any nucleotide sequence encoding a known or putative gene product.

- As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome. The term "locus" usually refers to the specific physical location of an endonuclease's target sequence on a chromosome. Such a 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". Also, 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. By "endogenous 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.

- By the expression "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

58 of an intervening sequence between two endonuclease-, rare-cutting endonuclease- or chimeric rare-cutting endonuclease-cutting sites. As another non-limiting example, 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. In this case, 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.

- By the expression "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.

- By "scarless re-ligation" is intended the perfect re-ligation event, without loss of genetic information (no insertion/deletion events) of the DNA broken ends through NHEJ process after the creation of a double-strand break event.

- By "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.

- By "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).

- By "chimeric rare-cutting endonuclease" is meant 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". A "chimeric rare-cutting endonuclease" according to the present invention which comprises more than two catalytic domains can be described as "multi- functional" or as "multi-functional meganuclease". As non-limiting examples, 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. As mentioned previously, 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. In other non-limiting examples, 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. In this definition are encompassed mutants, variants or derivatives of meganucleases (including monomers and single chain meganucleases), endonucleases, rare-cutting endonucleases or chimeric rare-cutting endonucleases.

- By "enhancer 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%. Non-limiting examples of such 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. - By "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

60 (http://wwwxhem.qmul.ac.uk/iubmb/enzyme/). In the scope of the present invention, 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. In a preferred embodiment of the present invention, 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.

- By "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". By "cleavase domain" is intended a protein domain whose catalytic activity generates a Double Strand Break (DSB) in a DNA target. By "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.

The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

As used above, the phrases "selected from the group consisting of," "chosen from," and the like include mixtures of the specified materials.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

61 2386

Examples Example 1

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. For the two N-terminal fusion scaffolds, 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. For both fusion scaffold types, the "free" end of the linker (i.e. onto which a polypeptide can be linked) is designed to be proximal to the DNA, as determined from models built using the l-Crel/DNA complex structures as a starting point (PDB id: lg9z). The two l-Crel N-terminal fusion scaffolds (l-Crel_NFSl = SEQ ID NO: 6 and l-Crel_NFS2 = SEQ ID NO: 7) and the single C-terminal fusion scaffold (l-Crel_CFSl = SEQ ID NO: 8) were tested in our 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]. Guided by biochemical and structural data, 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). As l-Crel is a homodimer, all fusion constructs contain two HNH domains (Figure 3A, where "enhancer domain" = the l-Hmul catalytic domain).

The activity of each "enhanced" meganuclease (eMega) was assessed using yeast assay described in International PCT Applications WO 2004/067736. To monitor the effect on activity resulting from the addition of the new enhancer domains, 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. In all cases, fusion proteins strongly enhanced cleavage of targets relative to the

62 activity of wild-type l-Crel (Table 3). In addition, the hHmuCre fusion hybrids were able to cleave "2NN" targets not cleaved by wild-type l-Crel at either 37°C (Table 4) or 30°C (Table 5).

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

63 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.

64 TACA - + +

TATA - + + +

TCCA - + + +

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.

Example 2

Using the truncated l-Cre_X scaffold (SEQ ID NO: 5) as a starting point, constructs were generated by adding variable length regions of the CFP1 peptide (SEQ ID NO: 112) to the C-terminus of the protein. As l-Crel is a homodimer, all fusion constructs contain two CFP1 peptides (Figure 3A, where "enhancer domain" = the CFP1 peptide domain).

The activity of each "enhanced" meganuclease (eMega) was assessed using yeast assay described in International PCT Applications WO 2004/067736 (see Example 1). To monitor the effect on activity resulting from the addition of the new enhancer domains, 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.

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72

Claims

A method for enhancing rare-cutting endonuclease efficiency at a genomic locus of interest in a cell comprising the steps of:

(i) identifying at said genomic locus of interest one DNA target sequence cleavable by one rare-cutting endonuclease;

(ii) engineering said rare-cutting endonuclease in order to create a chimeric rare-cutting endonuclease wherein said chimeric rare-cutting endonuclease is a fusion protein comprising at least a rare-cutting endonuclease-derived scaffold and at least one enhancer domain and wherein said chimeric rare-cutting endonuclease has an enhanced efficiency for said DNA target sequence relative to the efficiency of the starting rare-cutting endonuclease for said DNA target sequence;

(iii) contacting said DNA target sequence with said chimeric rare-cutting endonuclease for cleavage within the genomic locus of interest; thereby obtaining a cell in which rare-cutting endonuclease efficiency at a genomic locus of interest in a cell is enhanced.

The method according to claim 1 wherein said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold.

The method according to claim 1 wherein said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.

The method according to claim 1 wherein 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.

The method according to claim 1 wherein 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.

73 6) The method according to claim 5 wherein said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of l-Crel_NFSl (SEQ ID NO: 6); I- Crel_NFS2 (SEQ ID NO: 7) and l-Crel_CFSl (SEQ ID NO: 8).

7) The method according to claim 1 wherein said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of proteins listed in Table 1, a functional mutant, a variant or a derivative thereof.

8) The method according to claim 7 wherein said enhancer domain is l-Hmul, a functional mutant, a variant or a derivative thereof.

9) The method according to claim 1 wherein said chimeric rare-cutting endonuclease further comprises at least one peptidic linker between said rare-cutting endonuclease-derived scaffold and said at least one enhancer domain.

10) The method according to claim 9 wherein said peptidic linker sequence is selected from the group consisting of peptides listed in Table 2.

11) The method according to claim 1 wherein said chimeric rare-cutting endonuclease processes 2NN derivatives of said DNA target sequence that are not cleaved by wild-type rare-cutting endonuclease.

12) The method according to claim 1 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).

13) A method to create a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence comprising the steps of:

(i) Engineering a rare-cutting endonuclease-derived scaffold from a rare-cutting endonuclease capable to cleave said DNA target sequence;

(ii) determining or engineering an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease when fused to it;

(iii) optionally determining or engineering a peptidic linker to fuse said rare-cutting endonuclease-derived scaffold to said enhancer domain;

74 thereby obtaining a chimeric rare-cutting endonuclease with enhanced efficiency for a DNA target sequence.

14) The method according to claim 13 wherein said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold.

15) The method according to claim 13 wherein said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.

16) The method according to claim 13 wherein 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.

17) The method according to claim 13 wherein said rare-cutting endonuclease-derived scaffold is derived from l-Crel, a functional mutant of l-Crel, a variant of l-Crel or a derivative thereof.

18) The method according to claim 17 wherein said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of l-Crel_NFSl (SEQ ID NO: 6); I- Crel_NFS2 (SEQ ID NO: 7) and l-Crel_CFSl (SEQ ID NO: 8).

19) The method according to claim 1 wherein said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of proteins listed in Table 1, a functional mutant, a variant or a derivative thereof.

20) The method according to claim 19 wherein said enhancer domain is selected from the group consisting of l-Hmul, a functional mutant, a variant or a derivative thereof.

21) The method according to claim 20 wherein said chimeric rare-cutting endonuclease can comprise a peptidic linker selected from the group consisting of peptides listed in Table 2.

22) The method according to claim 13 wherein said chimeric rare-cutting endonuclease processes 2NN derivatives of said DNA target sequence that are not cleaved by wild-type rare-cutting endonuclease.

23) A chimeric rare-cutting endonuclease for a DNA target sequence comprising:

75 (i) a rare-cutting endonuclease-derived scaffold;

(ii) an enhancer domain wherein said enhancer domain enhances the efficiency of said rare-cutting endonuclease for said DNA target sequence when fused to it;

(iii) optionally a peptidic linker.

24) The chimeric rare-cutting endonuclease according to claim 23 wherein said enhancer domain is fused to the N-terminus part of said rare-cutting endonuclease-derived scaffold.

25) The chimeric rare-cutting endonuclease according to claim 23 wherein said enhancer domain is fused to the C-terminus part of said rare-cutting endonuclease-derived scaffold.

26) The chimeric rare-cutting endonuclease according to claim 23 wherein 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.

27) The chimeric rare-cutting endonuclease according to claim 23 wherein said rare-cutting endonuclease-derived scaffold is derived from l-Crel, a functional mutant of l-Crel, a variant of l-Crel or a derivative thereof.

28) The chimeric rare-cutting endonuclease according to claim 27 wherein said rare-cutting endonuclease-derived scaffold comprises a sequence selected from the group consisting of I- Crel_NFSl (SEQ ID NO: 6); l-Crel_NFS2 (SEQ ID NO: 7) and l-Crel_CFSl (SEQ ID NO: 8).

29) The chimeric rare-cutting endonuclease according to claim 23 wherein said enhancer domain consists of a protein domain derived from a protein selected from the group consisting of SEQ ID NOs: 10-66.

30) The chimeric rare-cutting endonuclease according to claim 23 wherein said enhancer domain is l-Hmul.

31) The chimeric rare-cutting endonuclease according to claim 23 wherein said peptidic linker sequence can be selected from the group consisting of SEQ ID NOs: 67-104

32) The chimeric rare-cutting endonuclease according to claim 23 wherein said chimeric rare- cutting endonuclease processes 2NN derivatives of said DNA target sequence that are not cleaved by wild-type rare-cutting endonuclease.

76 33) The chimeric rare-cutting endonuciease according to claim 23 wherein said chimeric rare- cutting endonuciease is selected from the group consisting of SEQ ID NO: 9 and SEQ ID NOs: 105-109.

34) A recombinant polynucleotide encoding a chimeric rare-cutting endonuciease according to claim 23.

35) A vector comprising a recombinant polynucleotide according to claim 34.

36) A composition comprising a chimeric rare-cutting endonuciease according to claim 23 and a carrier.

37) A pharmaceutical composition comprising a chimeric rare-cutting endonuciease according to claim 23 and a pharmaceutically active carrier. 38) A host cell which comprises a recombinant polynucleotide of claim 34.

39) A non-human transgenic animal which comprises a recombinant polynucleotide of claim 34.

40) A non-human transgenic animal which comprises a vector of claim 35.

41) A transgenic plant which comprises a recombinant polynucleotide of claim 34. 42) A transgenic plant which comprises a vector of claim 35.

43) A method of treatment of a genetic disease caused by a mutation in a specific double- stranded DNA target sequence in a gene comprising administering to a subject in need thereof an effective amount of a chimeric rare-cutting endonuciease according to claim 23.

44) A method for inserting a transgene into a specific double-stranded DNA target sequence of a genomic locus of a cell, tissue or non-human animal wherein at least one chimeric rare- cutting endonuciease according to claim 23 is introduced in said cell, tissue or non-human animal.

45) A kit comprising a chimeric rare-cutting endonuciease according to claim 23 and instructio for use in enhancing efficiency for a DNA target sequence.

77 46) A method for increasing targeted Homologous Recombination by using chimeric rare-cutting endonucleases according to claim 23.

A method for increasing targeted Homologous Recombination with less Non Homologous End-joining by using chimeric rare-cutting endonucleases according to claim 23 wherein at least one enhancer domain is a catalytically active nickase domain.

48) A method for increasing excision of a single-strand of DNA spanning the starting rare-cutting endonuclease binding region by using chimeric rare-cutting endonucleases according to claim 23 wherein:

(i) one enhancer domain is a catalytically active nickase domain;

(ii) one enhancer domain is a catalytically active cleavase domain;

the rare-cutting endonuclease-derived scaffold is an inactive rare-cutting endonuclease scaffold which serves as a scaffold for targeted DNA binding.

49) A chimeric rare-cutting endonuclease for a DNA target sequence comprising:

(i) a rare-cutting endonuclease-derived scaffold;

(ii) an enhancer domain fused to a rare-cutting endonuclease; and optionally

(iii) a peptidic linker.

50) A method for enhancing processing of a nucleic acid sequence compared to the processing of a parent rare-cutting endonuclease comprising:

(i) identifying one DNA target sequence recognized by one rare-cutting endonuclease; (ii) producing a chimeric rare-cutting endonuclease by fusing at least a rare-cutting endonuclease-derived scaffold with at least one enhancer domain;

(iii) selecting a chimeric rare-cutting endonuclease with enhanced efficiency to process a nucleic acid sequence compared to the parent rare-cutting endonuclease;

(iv) contacting a nucleic acid containing said DNA target sequence with said chimeric rare- cutting endonuclease for a time an under conditions sufficient for processing of said nucleic acid.

78