SG193850A1 - Meganuclease variants cleaving a dna target sequence from a glutamine synthetase gene and uses thereof - Google Patents

Abstract

86 Abstract MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM A GLUTAMINE SYNTHETASE GENE AND USES THEREOFAn I-Crel variant, wherein one of the two I-Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 28 to 40 and 44 to 77 of I-Crel, said variant being able to cleave a DNA target sequence from the Glutamine Synthetase gene. Use of said variant and derived products for improving expression system for the production of recombinant protein.No suitable fig.

Description

MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE

FROM A GLUTAMINE SYNTHETASE GENE

AND USES THEREOF :

The invention relates to a meganuclease variant cleaving a DNA . target sequence from a Glutamine Synthetase (GS) gene, to a vector encoding said variant, to a‘cell, an animal or a plant modified-by said vector-and to the use of said meganuclease variant and derived products for genome engineering and for in vivo and ex vivo (gene cell therapy) genome therapy.

Glutamine Synthetase (GS), also called glutamate-ammonia ligase (GLUL), is a universal housekeeping enzyme responsible for the biosynthesis of glutamine from glutamate and ammonium, using the hydrolysis of ATP to ADP and : phosphate to drive the reaction. As such, it represents an important link between the

Krebs cycle and amino acid metabolism (Meister ef a/., 1980, Glutamine metabolism, enzymology and regulation, Academic Press, N.Y., p.1-40 and 319-329). This enzymatic reaction is the pathway for glutamine formation in mammalian cells. In the absence of glutamine in the growth medium, the GS enzyme plays an essential role in the survival of mammalian cells in culture. Glutamine Synthetase is encoded by one of : the oldest existing and functioning genes in the history of gene evolution and can be regarded as a key enzyme in the metabolism of prokaryotes and eukaryotes (Kumada et al, 2003, PNAS USA, 90: 3009-3013). Given its biological function, the GS gene is used as a positive selection marker for genome engineering (targeted and random gene manipulations).

GS is found at low levels (0.01% - 0.1% of soluble protein) in most higher vertebrate cells and is found at higher levels (>1% of total protein) in certain specialized cell types such as hepatocytes, adipocytes and glial cells (Tiemeier ef al, 1972, J. Biol. Chem., 247: 2272-2277; Gebhardt et al, 1983, EMBO J., 2: 567-570;

Miller et al., 1978, PNAS USA, 75:1418-1422; Linser ef al., 1979, PNAS USA, 76: 6476-6480). A variety of regulatory signals affect GS levels within cells, for instance glucocorticoid steroids and cAMP, and glutamine in a culture medium appears to regulate GS levels post-translationally (Milman et al., 1975, J. Biol. Chem., 250: 1393-1399; Arad et al., 1976, Cell, 8:59-101) via ADP ribosylation.

Some mammalian cell lines, such as mouse myeloma lines, do not express sufficient GS to survive without added glutamine. With these cell lines, a transfected GS gene can function as a selectable marker by permitting growth in a glutamine-free medium. Other cell lines, such as Chinese Hamster Ovary (CHO) cell lines, express sufficient GS to survive without exogenous glutamine. In these cases, a

GS inhibitor, such as methionine sulphoximine (Msx), can be used to inhibit endogenous GS activity such that only transfectants with additional GS activity can survive.

Mammalian cells are attractive for protein production since such proteins are generally correctly folded, appropriately modified and completely functional, often in marked contrast to proteins expressed in bacterial cells.

A mammalian expression system, named GS System™, has been developed by Lonza Biologics using CHO-K1 cells for the production of a desired protein. CHO-K1 cells produce endogenous GS, but they can be used, to produce stable cell lines by transfecting in a GS gene and using a glutamine-free medium plus

Msx (at sufficient levels to inhibit the endogenous enzyme) to provide selection pressure, along with the transfection of a gene of interest. :

The GS System™ has been used to produce a wide variety of recombinant proteins, in particular therapeutic products which have been approved by regulatory authorities. Currently there are over 50 products in clinical trials and 5 products in-market that use the GS System™, such as Zenapax® (Roche) and Synagis® (MedImmune).

Nevertheless, the use of GS inhibitor in order to inhibit GS endogenous expression is not entirely satisfactory as a residual expression remains.

This problem could be overcome by inactivating directly the endogenous GS gene.

This inactivation could be achieved by using a site-specific endonuclease such as meganucleases which are able to create a DNA double-strand break (DSB) and cleave unique sites in living cells. This cleavage could be then repaired by Homologous

Recombination (Figure 3A) or Non Homologous End Joining (NHEJ) (Figure 3B).

Thus, an artificial meganuclease targeting the GS gene could be used to inactivate the

GS gene.

Glutamine Synthetase is also ubiquitously expressed in the human organism with high concentrations in liver, brain and muscular tissues (Haussinger D et al., 1984, Glutamate Metabolism in Mammalian Tissues. Berlin: Springer Verlag,

3-15). GS plays a major role in ammonia and glutamate detoxification, interorgan nitrogen flux, pH homeostasis and cell signaling (Haussinger D, 1998, Adv Enzymol

RAMB 72: 43-86). Inherited systemic deficiency of glutamine based on a defect of

Glutamine Synthetase has been described (Haberle er al, 2006, J Inherit Metab Dis, 29, 352-358) in two newborns with an early fatal course of disease. Glutamine was largely absent in their serum, urine and cerebrospinal fluid. Homozygous mutations in exon 7 of the Glutamine Synthetase gene were detected in both of the patients. One patient carried -an arginine324-to-cysteine substitution (R324C) and the other an arginine341-to-cysteine substitution . (R341C). Glutamine Synthetase Enzymatic investigations confirmed that these mutations lead to a severely reduced Glutamine

Synthetase activity.

Targeted homologous recombination should allow for the precise correction of mutations in situ (Figure 3C). Therefore, an artificial meganuclease targeting the GS gene could be used for repairing the mutations associated with inherited systemic deficiency of glutamine.

Homologous recombination (HR), is a very conserved DNA maintenance pathway involved in the repair of DNA double-strand breaks (DSBs) and other DNA lesions (Rothstein, Methods Enzymol., 1983, 101, 202-211; Paques ef al.,

Microbiol Mol Biol Rev, 1999, 63, 349-404; Sung ef gl., Nat. Rev. Mol. Cell. Biol, 2006, 7, 739-750) but it also underlies many biological phenomenon, such as the . meiotic reassortiment of alleles in meiosis (Roeder, Genes Dev., 1997, 11, 2600- 2621), mating type interconversion in yeast (Haber, Annu. Rev. Genet., 1998, 32, 561- 599), and the “homing” of class I introns and inteins to novel alleles. HR usually promotes the exchange of genetic information between endogenous sequences, but in gene targeting experiments, it is used to promote exchange between an endogenous chromosomal sequence and an exogenous DNA construct. Basically, a DNA sharing homology with the targeted sequence is introduced into the cell’s nucleus, and the endogenous homologous recombination machinery provides for the next steps (Figure 3C).

Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M.R., Science, 1989, 244, 1288-1292, Smithies, O.,

Nature Medicine, 2001, 7, 1083-1086) or knock-in exogenous sequences in the chromosome. It can also be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases. However, this application is in fact difficult, due to the low efficiency of the process (10° to 10” of transfected cells). .

One of several strategies to enhance the efficiency of recombination is to deliver a DNA double-strand break in the targeted locus, using meganucleases.

Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (Thierry, A. and B. Dujon, Nucleic Acids Res., 1992, 20, 5625-5631). They : can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al, Nucleic Acids Res., 1993, 21, 5034-5040 ; Rouet ef al., Mol. Cell. Biol., 1994, 14, 8096-8106 ; Choulika ef al.,

Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta ef al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 5055-5060 ; Sargent er al, Mol. Cell. Biol., 1997, 17, 267-277; Cohen-

Tannoudji ef al., Mol. Cell. Biol., 1998, 18, 1444-1448 ; Donoho, ef al, Mol. Cell.

Biol, 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101). Such meganucleases could be used to correct mutations responsible for monogenic inherited diseases.

The most accurate way to correct a genetic defect is to use a repair matrix with a non mutated copy of the gene, resulting in a reversion of the mutation.

However, the efficiency of gene correction decreases as the distance between the mutation and the DSB grows, with a five-fold decrease at a distance of 200 bp.

Therefore, a given meganuclease can be used to correct only mutations in the vicinity of its DNA target (Figure 3C).

An alternative, termed “exon knock-in” is featured in Figure 3D. In this case, 2 meganuclease cleaving in the 5° part of the gene can be used to knock-in functional exonic sequences upstream of the deleterious mutation. Although this method places the transgene in its regular location, it also results in duplication of exons, whose long term impact remains to be evaluated. In addition, should naturally cis-acting elements be located in an intron downstream of the cleavage, their immediate environment would be modified and their proper function would also need to be explored. However, this method has a tremendous advantage: a single meganuclease could be used for many different mutations downstream of the meganuclease cleavage site.

However, although several hundreds of natural meganucleases, also referred to as “homing endonucleases” have been identified (Chevalier et al, 2001, 5 Nucleic Acids Res., 29,3757 -3774), the repertoire of cleavable sequences is too limited to address the complexity of the genomes, and for example, there is no cleavable site in the GS gene. Theoretically, the making of artificial sequence specific endonucleases with chosen specificities could alleviate this limit. Therefore, the making of meganucleases with tailored specificities is under intense investigation.

Recently, fusion of Zinc-Finger Proteins (ZFPs) with the catalytic domain of the Fokl, a class IIS restriction endonuclease, were used to make functional sequence-specific endonucleases (Smith ef al, Nucleic Acids Res., 1999, 27, 674-681;

Bibikova et al., Mol. Cell. Biol, 2001, 21, 289-297 ; Bibikova ef al., Genetics, 2002, 161, 1169-1175 ; Bibikova et al., Science, 2003, 300, 764 ; Porteus, MH. and D.

Baltimore, Science, 2003, 300, 763-; Alwin er al, Mol. Ther., 2005, 12, 610-617;

Umov et al., Nature, 2005, 435, 646-651; Porteus, M.H., Mol. Ther., 2006, 13, 438- 446).

The binding specificity of Cys2-His2 type Zinc-Finger Proteins, is easy to manipulate, probably because they represent a simple (specificity driven by essentially four residues per finger), and modular system (Pabo ef al, Annu. Rev.

Biochem., 2001, 70, 313-340 ; Jamieson ef al., Nat. Rev. Drug Discov., 2003, 2, 361- 368. Studies from the Pabo (Rebar, E.J. and C.O. Pabo, Science, 1994, 263, 671-673 ;

Kim, J.S. and C.O. Pabo, Proc. Natl. Acad. Sci. U S A, 1998, 95, 2812-2817), Klug (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167 ; Isalan

M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) and Barbas (Choo, Y. and A.

Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167; Isalan M. and A. Klug,

Nat. Biotechnol, 2001, 19, 656-660) laboratories resulted in a large repertoire of } novel artificial ZFPs, able to bind most G’/ANNG/ANNG/ANN sequences. "Nevertheless, ZFPs might have their limitations, especially for applications requiring a very high level of specificity, such as therapeutic applications.

The Fokl nuclease activity in fusion acts as a dimer, but it was recently shown that it could cleave DNA when only one out of the two monomers was bound to DNA, or when the two monomers were bound to two distant DNA sequences (Catto ef al.,

Nucleic Acids Res., 2006, 34, 1711-1720). Thus, specificity might be very degenerate, as illustrated by toxicity in mammalian cells (Porteus, M.H. and D. Baltimore,

Science, 2003, 300, 763) and Drosophila (Bibikova ef al., Genetics, 2002, 161, 1169- 1175; Bibikova ef al., Science, 2003, 300, 764-.).

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, B.S. and B.L.

Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). These proteins are encoded by mobile genetic elements which propagate by a process called “homing” the endonuclease cleaves a cognate allele from which the mobile element is absent, ‘thereby stimulating a homologous recombination event that duplicates the mobile

DNA into the recipient locus. 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 four major families. The LAGLIDADG family, named Co after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Seven 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 (Figure 1A). The catalytic core is flanked by two DNA-binding domains with a perfect two- fold symmetry for homodimers such as I-Crel (Chevalier, et al., Nat. Struct. Biol., 2001, 8, 312-316) , I-Msol (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269) and I- : Ceul (Spiegel et al., Structure, 2006, 14, 869-880} and with a pseudo symmetry for monomers such as I-Scel (Moure et al, J. Mol. Biol., 2003, 334, 685-69, I-Dmol (Silva et al, J. Mol. Biol., 1999, 286, 1123-1136) or [-Anil (Bolduc ef al., Genes Dev., 2003, 17, 2875-2888). 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 appoappc folds, sitting on the DNA major groove. Other domains can be found, for example in inteins such as PI-Pful (Ichiyanagi et al, J. Mol. Biol., 2000, 300, 889-901) and PI- ‘Scel (Moure ef al, Nat. Struct. Biol, 2002, 9; 764-770), whose protein splicing domain is also involved in DNA binding.

The making of functional chimeric meganucleases, by fusing the N- terminal I-Dmol domain with an I-Crel monomer (Chevalier et al., Mol. Cell., 2002, 10, 895-905 ; Epinat ef al., Nucleic Acids Res, 2003, 31, 2952-62; International PCT

Applications WO 03/078619 and WO 2004/031346) have demonstrated the plasticity of LAGLIDADG proteins.

Different groups have also used a semi-rational approach to locally alter the specificity of the I-Crel (Seligman ef al., Genetics, 1997, 147, 1653-1664;

Sussman ef al, J. Mol. Biol, 2004, 342, 31-41; International PCT Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO 2007/049156; Arnould ef al, J. Mol. Biol., 2006, 355, 443-458; Rosen ef al., Nucleic Acids Res., 2006, 34, 4791-4800 ; Smith ef al., Nucleic Acids Res., 2006, 34, e149), I-Scel (Doyon et al., J.

Am. Chem. Soc., 2006, 128, 2477-2484), PI-Scel (Gimble et al., J. Mol. Biol., 2003, 334, 993-1008 ) and I-Msol (Ashworth ef al., Nature, 2006, 441, 656-659).

In addition, hundreds of I-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 I-Crel were mutagenized and a collection of variants with altered specificity at positions + 3 to 5 of the DNA target (SNNN DNA target) were identified by screening (International PCT Applications WO 2006/097784 and WO 2006/097853; Amould et al., J. Mol. Biol., 2006, 355, 443-458; Smith et al, Nucleic Acids Res., 2006, 34, el49). - Residues K28, N30 and Q38, N30, Y33 and Q38 or K28, Y33, Q38 and S40 of I-Crel were mutagenized and a collection of variants with altered speci- ficity at positions * 8 to 10 of the DNA target. (10NNN DNA target) were identified by screening (Smith ef al, Nucleic Acids Res., 2006, 34, e149; International PCT

Applications WO 2007/060495 and WO 2007/049156).

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 (Amould ef al., precited,

Intemational PCT Applications WO 2006/097854 and WO 2007/034262), as ’ : 5 illustrated on Figure 1B.

Furthermore, residues 28 to 40 and 44 to 77 of [-Crel were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease half-site (Smith et al. Nucleic Acids Res., 2006, 34, e149; International

PCT Applications WO 2007/049095 and WO 2007/057781).

The combination of mutations from the two subdomains of 1-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 et al, Nucleic Acids Res., 2006, 34, e149; Intemational PCT

Applications WO 2007/049095 and WO 2007/057781). :

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 et al, Nucleic Acids Res., 2003, 31, 2952- 2962; Chames ef al., Nucleic Acids Res., 2005, 33, e178, and Amould ef al., J. Mol.

Biol., 2006, 355, 443-458. 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, as illustrated on Figure 1C. In a first step, couples of novel meganucleases are : combined in new molecules (“half-meganucleases™) cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such “half- meganucleases” can result in a heterodimeric species cleaving the target of interest.

The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from the human RAGI1, XPC and HPRT genes have been described in Smith ef al. (Nucleic Acids Res., 2006, 34, e149), Arnould er al., (J. Mol. Biol., 2007, 371, 49-65), and WO2008/059382 respectively.

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

However, even though the base-pairs 1 and +2 do not display any contact with the protein, it has been shown that these positions are not devoid of content information (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269), especially for the base-pair %1 and could be a source of additional substrate specificity (Argast et al, J. Mol. Biol., 1998, 280, 345-353; Jurica et al., Mol. Cell., 1998, 2, 469-476;

Chevalier, B.S. and B.L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). In vitro selection of cleavable I-Crel targets (Argast ef al., precited) randomly mutagenized, revealed the importance of these four base-pairs on protein binding and cleavage activity. It has been suggested that the network of ordered water molecules found in the active site was important for positioning the DNA target (Chevalier et al,

Biochemistry, 2004, 43, 14015-14026). In addition, the extensive conformational changes that appear in this region upon I-Crel binding suggest that the four central nucleotides could contribute to the substrate specificity, possibly by sequence dependent conformational preferences (Chevalier et al., 2003, precited).

Thus, it was not clear if variants identified on 10NNN and 5NNN

DNA targets as homodimers cleaving a palindromic sequence with the four central nucleotides being gtac, would allow the design of new endonucleases that would cleave targets containing changes in the four central nucleotides.

The Inventor has identified a series of DNA targets in the GS gene : that could be cleaved by I-Crel variants (figures 18 to 20). The combinatorial approach, as illustrated in Figure 1D was used to entirely redesign the DNA binding domain of the I-Crel protein and thereby engineer novel meganucleases with fully engineered specificity, to cleave one DNA target (GSCHOL1). The GSCHOI target is present in both mouse (Figure 2A) and Chinese Hamster (Criteculus griseus; Figure 2B) GS genes and differs from the I-Crel C1221 22 bp palindromic site by 15 nucleotides including two (positions +1, +2) out of the four central nucleotides (Figure

In a first step, couples of novel meganucleases are combined in new molecules (“half-meganucleases™) cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such “half-meganucleases” can _ result in a heterodimeric species cleaving the target of interest. The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from the human RAG! gene has been described previously in Smith ef al., Nucleic Acids Res., 2006, 34, e149.

Even though the combined variants were initially identified towards nucleotides 10NNN and SNNN respectively, and a strong impact of the four central 10" nucleotides of the target on the activity of the engineered meganuclease was observed, functional meganucleases with a profound change in specificity were selected.

Furthermore, the activity of the engineered protein could be significantly improved by random and/or site-directed mutagenesis and screening, to compare with the activity of the I-Crel protein.

These I-Crel variants which are able to cleave a genomic DNA target from the GS gene can be used for inactivating the GS locus (knock-out and knock-in) (Figure 3A and 3B), thus allowing GS to be used as a selectable marker for genome engineering at any locus, for example for making transgenic animals and recombinant cell lines. In addition, these I-Crel variants could be used for repairing the GS mutations associated with inherited systemic deficiency of glutamine (Figure 3C and 3D).

The invention relates to an [-Crel variant wherein at least one of the two [-Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 28 to 40 and 44 to 77 of [-Crel, and is able to cleave a DNA target sequence from the GS gene.

The cleavage activity of the variant according to the invention may be measured by any well-known, ir vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736; Epinat ef al,

Nucleic Acids Res., 2003, 31, 2952-2962; Chames ef al., Nucleic Acids Res., 2005, 33, el 78; Amould et al., J. Mol. Biol., 2006, 355, 443-458, and Arnould ef al., I. Mol.

Biol., 2007, 371, 49-65.For example, the cleavage activity of the variant of the invention may be measured by a direct repeat recombination assay, in yeast or - mammalian cells, using a reporter vector. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic (non-palindromic) DNA target sequence within the intervening sequence, cloned in yeast or in a mammalian expression vector, Usually, the genomic DNA target sequence comprises one different half of each (palindromic or pseudo-palindromic) parent homodimeric [-Crel meganuclease target sequence. Expression of the heterodimeric variant results in a functional endonuclease which is able to cleave the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene, whose expression can be monitored by an appropriate assay. The cleavage activity of the variant against the genomic DNA target may be compared to wild type I-Crel or I-Scel activity against their natural target.

Definitions - Amino acid residues in a polypeptide sequcnce are designated herein according to the one-letter code, in which, for example, Q means Gln or

Glutamine residue, R means Arg or. Arginine residue and D means Asp or Aspartic acid residue, : © - Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or ¢, w represents a or t, m represents a or ¢, y repre- sents t or ¢ (pyrimidine nucleotides), d represents g, a or t, v represents g, a orc, b represents g, t or ¢, h represents a, t or ¢, and n represents g, a, t or c. - by “meganuclease”, is intended an endonuclease having a double- stranded DNA target sequence of 12 to 45 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 which interacts with the other half of the

DNA target to form a functional meganuclease able to cleave said DNA target.

- by “meganuclease variant” or “variant” is intended a meganuclease obtained by replacement of at least one residue in the amino acid sequence of the wild-type meganuclease (natural meganuclease) with a different amino acid. - by “functional variant” is intended a variant which is able to cleave a DNA target sequence, preferably said target is a new target which is not cleaved by the parent meganuclease. For example, such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. - by “I-Crel” is intended the wild-type [-Crel having the sequence of pdb accession code 1g9y, corresponding to the sequence SEQ ID NO: 1 in the sequence listing. - by “I-Crel variant with novel specificity” is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease. The terms “novel specificity”, “modified specificity”, “novel cleavage specificity”, “novel 16 substrate specificity” which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence. - by “I-Crel site” is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by [-Crel. I-Crel sites include the wild-type (natural) non- palindromic I-Crel homing site and the derived palindromic sequences such as the sequence 5°- t)3C.112.1029238.7C 48-51.4C-38.2L18+1C42843244C+58sstrtagtiotr 1084112412 (SEQ ID NQ: 2), also called C1221 (Figure 4). - by "domain" or "core domain" is intended the “LAGLIDADG homing endonuclease core domain” which is the characteristic a; 3;8202p3B4cta 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 (B1B2B3fB4) 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 endonuclease I-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 endo- nuclease 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 (3p: or,B3p4) which are connected by a loop or a turn, : - by “single-chain meganuclease”, “single-chain chimeric meganu- clease”, “single-chain meganuclease derivative”, “single-chain chimeric meganuclease derivative” or “single-chain derivative” is intended a meganuclease comprising two

LAGLIDADG homing endonuclease domains or core domains linked by a peptidic spacer. The single-chain meganuclease is able to cleave a chimeric DNA target sequence comprising one different half of each parent meganuclease target sequence. - by “DNA target”, “DNA target sequence”, “target sequence”, “target-site”, “target”, “site”; "site of interest"; “recognition site”, “recognition sequence”, “homing recognition site”, “homing site”, “cleavage site” is intended a 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a

LAGLIDADG homing endonuclease such as I-Crel, or a variant, or a single-chain chimeric meganuclease derived from I-Crel. These terms refer to a distinct DNA location, preferably a genomic location, at which a double stranded break (cleavage) is to be induced by the meganuclease. The DNA target is defined by the 5° to 3’ sequence of one strand of the double-stranded polynucleotide, as indicate above for

C1221. Cleavage of the DNA target occurs at the nucleotides at positions +2 and -2, respectively for the sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an I-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target. - by "DNA target half-site", “half cleavage site” or half-site” is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain. - by "chimeric DNA target" or "hybrid DNA target" is intended the fusion of different halves of two parent meganuclease target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target). - by “GS gene” is intended a Glutamine Synthetase or Glutamate-

Ammonia Ligase (GLUL) gene, preferably the GS gene of a vertebrate, more preferably the GS gene of a mammal such as human, mouse and Chinese Hamster (Criteculus griseus) GS genes. GS gene sequences are available in sequence databases, such as the NCBI/GenBank database. The human GS gene sequence (9282 bp; SEQ ID NO: 272) is available under accession number NC _000001.9 (reverse complement of positions 180618292 to 180627573). The mouse GS gene sequence (9770 bp; SEQ ID NO: 3) is available under accession number NC_000067.5 (reverse : complement of positions 155747075 to 155756844). Both genes have 7 exons. The mouse GS gene is illustrated by figure 2A (Exon 1 (positions 1 to 115), Exon 2 (positions 2990 to 3168), Exon 3 (positions 4593 to 4754), Exon 4 (positions 6405 to 6551), Exon 5 (positions 7076 to 7203), Exon 6 (positions 7342 to 7541) and Exon 7 (positions 7920 to 9770)). The human GS gene comprises: Exon 1 (positions 1 to 137), Exon 2 (positions 3066 to 3244), Exon 3 (positions 4524 to 4685), Exon 4 (positions 5414 to 5560), Exon 5 (positions 5929 to 6056), Exon 6 (positions 6260 to 6459) and Exon 7 (positions 7093 to 9282). The ORF which is from the beginning of

Exon 2 (positions 3003 (mouse GS)) or 3079 (human GS)) to the beginning of Exon 7 (positions 8238 (mouse GS) or 7411 (human GS)), is flanked by long untranslated regions, respectively at the 5° and 3’ end. The mouse gene is transcribed into a 2782 bp mRNA (GenBank NM _008131) containing the GS ORF from positions 129 to 1250. The Chinese Hamster (Criteculus griseus) GS mRNA is a 1421bp sequence (accession number GenBank X03495) containing the GS ORF from positions 147 to 1268 (Figure 2B). - by “DNA target sequence from the GS pene”, “genomic DNA target sequence”, “ genomic DNA cleavage site”, “genomic DNA target” or “genomic © target” is intended a 20 to 24 bp sequence of a GS gene as defined above, which is recognized and cleaved by a meganuclease variant or a single-chain chimeric meganuclease derivative. - by "vector" is intended a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

- by "homologous" is intended a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95 % identity, preferably 97 % identity and more prefera- bly 99 %. - "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or

BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. - 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.

The variant according to the present invention may be a homodimer or a heterodimer. Preferably, both monomers of the heterodimer are mutated at positions 28 to 40 and/or 44 to 77. More preferably, both monomers have different substitutions both at positions 28 to 40 and 44 to 77 of [-Crel. ,

In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of I-Crel are at positions 44, 68, 70, 75 and/or 77.

In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 28 to 40 of I-Crel are at positions 28, 30, 32, 33, 38 and/or 40.

In another preferred embodiment of said variant, it comprises one or more mutations at positions of other amino acid residues that contact the DNA target sequence or interact with the DNA backbone or with the nucleotide bases, directly or via a water molecule; these residues are well-known in the art (Jurica ef al., Molecular

Cell, 1998, 2, 469-476; Chevalier ef al, J. Mol. Biol, 2003, 329, 253-269). In particular, additional substitutions may be introduced at positions contacting the ~ phosphate backbone, for example in the final C-terminal loop (positions 137 to 143;

Preto et al, Nucleic Acids Res., Epub 22 April 2007). Preferably said residues are involved in binding and cleavage of said DNA cleavage site. More preferably, said residues are at positions 138, 139, 142 or 143 of I-Crel. Two residues may be mutated in one variant provided that each mutation is in a different pair of residues chosen from the pair of residues at positions 138 and 139 and the pair of residues at positions 142 and 143. The mutations which are introduced modify the interaction(s) of said amino acid(s) of the final C-terminal loop with the phosphate backbone of the I-Crel site. Preferably, the residue at position 138 or 139 is substituted by a hydrophobic amino acid to avoid the formation of hydrogen bonds with the phosphate backbone of the DNA cleavage site. For example, the residue at position 138 is substituted by an alanine or the residue at position 139 is substituted by a methionine. The residue at position 142 or 143 is advantageously substituted by a small amino acid, for example a glycine, to decrease the size of the side chains of these amino acid residues. More, preferably, said substitution in the final C-terminal loop modify the specificity of the variant towards the nucleotide at positions + 1 to 2, £ 6 to 7 and/or + 11 to 12 of the I- Crel site.

In.another preferred embodiment of said variant, it comprises one or more additional mutations that improve the binding and/or the cleavage properties of the variant towards the DNA target sequence from the GS gene.

The additional residues which are mutated may be on the entire I- Crel sequence, and in particular in the C-terminal half of I-Crel (positions 80 to 163).

Both I-Crel monomers are advantageously mutated; the mutation(s) in each monomer may be identical or different. For example, the variant comprises one or more additional substitutions at positions: 2, 3, 6, 7, 12, 19, 24, 35, 39, 43, 45, 47, 50, 54, 57, 59, 60, 64, 66, 80, 87, 92, 96, 105, 107, 110, 114, 117, 118, 119, 120, 125, 129, 132,137, 139, 153, 154, 160 and 161. Said substitutions are advantageously selected from the group consisting of: N28, T3A, N6K, K7E, YI2H, GI9S, GI9A, 124V,

F35L, L39V, F43L, V45L, V45M, Q47K, Q50R, F54L, K57E, V594A, D60Y, V64A,

Y66H, E80K, F87L, F87I, Q92R, K96R, V105A, KI107R, E110V, S114F, S114P,

E117V, S118T, P119L, DI120A, DI120E, V125I, VI129A, 1132V, D137N, D137Y,

K139R, D153N, S154G, K160R, S161P and S161T. More preferably, the variant comprises at least one substitution selected from the group consisting of: G19S, F54L,

E80K, F87L, VI05A and I132V. The variant may also comprise additional residues at the C-terminus. For example a glycine (G) and/or a proline (P) residue may be inserted at positions 164 and 165 of 1-Crel, respectively.

According to a more preferred embodiment of said variant, said additional mutation further impairs the formation of a functional homodimer. More preferably, said mutation is the G19S mutation. The G19S mutation is advantageously introduced in one of the two monomers of a heterodimeric I-Crel variant, so as to obtain a meganuclease having enhanced cleavage activity and enhanced cleavage : specificity. In addition, to enhance the cleavage specificity further, the other monomer may carry a distinct mutation that impairs the formation of a functional homodimer or favors the formation of the heterodimer.

In another preferred embodiment of said variant, said substitutions - are replacement of the initial amino acids with amino acids selected from the group consisting of: A, D,E,G,H,K,N,P,Q,R,S, T,Y,C,V,L,M,F, I and W.

The variant of the invention may be’ derived from the wild-type I-

Creel (SEQ ID NO: 1) or an I-Crel scaffold protein having at least 85 % identity, preferably at least 90 % identity, more preferably at least 95 % identity with SEQ ID

NO: 1, such as the scaffold called I-Crel N75 (167 amino acids; SEQ ID NO: 4) having the insertion of an alanine at position 2, and the insertion of AAD at the C- terminus (positions 164 to 166) of the I-Crel sequence.

In addition, the variants of the invention may include one or more residues inserted at the NH; terminus and/or COOH terminus of the sequence. For example, a tag (epitope or polyhistidine sequence) is introduced at the NH; terminus and/or COOH terminus; said tag is useful for the detection and/or the purification of said variant. The variant may also comprise a nuclear localization signal (NLS); said

NLS is useful for the importation of said variant into the cell nucleus. The NLS may be inserted just after the first methionine of the variant or just after an N-terminal tag.

The variant according to the present invention may be a homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence.

Alternatively, said variant is a heterodimer, resulting from the association of a first and a second monomer having different substitutions at positions 28 to 40 and 44 to 77 of I-Crel, said heterodimer being able to cleave a non- palindromic DNA target sequence from the GS gene.

The DNA target sequences which are cleaved by the [-Crel variants are present in at least one mammalian GS gene selected from the group consisting of the human, mouse and/or Chinese Hamster (Crifeculus griseus) GS genes. The DNA target sequences are situated in the GS ORF and these sequences cover all the GS

ORF (figures 18 to 20).

For example, the DNA target sequences SEQ ID NO: 5 to 28 (figure 18) are present in the human GS gene. The DNA target sequences SEQ ID NO: 19 and 29 to 48 are present in the mouse GS gene (figure 19). The DNA target sequences

SEQ ID NO: 19, 29, 30, 34, 46, 47 and 49 to 60 are present in the Chinese Hamster

GS gene (figure 20).

The DNA target sequence SEQ ID NO: 19 is present in the human, . mouse and Chinese Hamster GS genes. Therefore, the I-Crel variants which cleave the DNA target sequence SEQ ID NO: 19 are able to induce a site-specific modification in the human, mouse and Chinese Hamster GS genes. The DNA target sequences SEQ ID NO: 29, 30, 34, 46 and 47 are present in both mouse and Chinese

Hamster GS genes. Therefore, the I-Crel variants which cleave the DNA target sequences SEQ ID NO: 29, 30, 34, 46 and 47 are able to induce a site-specific modification in the mouse and Chinese Hamster GS genes.

In addition, the human, mouse and Chinese. Hamster DNA target sequences SEQ ID NO: 7, 31 and 49 have sequence identity at the nucleotide positions + 3 to 5 and x 8 to 10. Therefore, the I-Crel variants which cleave the DNA target sequence SEQ ID NO: 49 are able to induce a site-specific modification in the

Chinese Hamster and for some of them, also in the human and/or mouse GS gene.

Examples of heterodimeric variants which cleave each DNA target are presented in figures 18 to 20 and Tables I to III.

Table I: Sequence of heterodimeric I-Crel variants cleaving a

DNA target from the human GS gene

Sequence Setuence (SEQ ID NO: 61 to 84) (SEQ ID NO: 85 to 108)

Ten mma

KTSGQS/VERNR+KE) KRTNQQYORSRT

Eom egmee [

KNGCAS/ARSNR KNSRDR/YASRI ay || 1

KRTCQT/KYSEV KRTNQQ/LRNNH+KBD erm me

KNSCASINTSRY KNSTASIMERNR aE | Sem [

KNSRNQ/RYSEV ENSRRK/NRSRY

Gein meses [|e

KNTCQSALRANV KYTCQS/DYSSR

EE LR

KNHHQS/NQSSY ’ KNSEQE/AYSYK

Er Jee 1

KRCCQE/KTTNI KNRDQS/ARSRL

BESET elev

KRSYQS/QESRR KSSHKS/INYSRV heme mmm

KNSTQS/ARSER KNSPQQ/KYSEV

Eee Semmes

KDSRTS/RRSND KNCCHS/RRSND ee a [vw

KNHHQS/YYSST KTSGQS/QRSYR

Ere een

KTSGQSKRSRR KNSRAQINRSRY a Jammer

KNSYQS/KYSNQ ENSRRK/KYSQN

Emm

KSSHKS/IRSNR KNDYCS/KESDR

Seem ee yo

KNSTQT/QNSQR KNSRAQIAQNNI

KNSROR/IRCNR KRANGE/ARSER

KNSPKSNKHNI RNSAYQIDYSSR

KNSCAS/IRANR KNTCQS/QRSHY i

Ee meen | a

KNTYWSIARSRL KNSRNQ/YSSSD

EET mee a

KDSRSS/YRSDV KNTYWS/DYSSR

KTSGQS/KRSDK NNSSRKJAYSRI

KNSRNQ/SRSYT KDSRQS/KESDR

KNSCAS/ARSER KRSRQS/GYRNI * the underlined variants can cleave the identical target found in the GS gene of another species.

Table II: Sequence of heterodimeric I-Crel variants cleaving a

DNA target from the mouse GS gene 5001055 rn | oa Ti nr 55

SEQ ID NO; 109 to 123, 75, 124 to 128) | (SEQ ID NO; 129 to 146, 99, 147 to 151

Ey

KNCCHS/KYSNI KRSYQSTYSRT

KRGYQS/RHRDI :

J0R 33R 38E 44D 68Y 70S 75Y 77Q KRGYQS/KHRDI 2

KRSRES/DYSYQ 30R 32G 44K 68N

KRCYQS/RHRDI

Frit lea CO I

KHHCQT/RYSEV KRTNQQ/IRCNR

SEE pe | | T

KNSRAQ/RYSER KNSHAS/VERNR +K80

KRGRQA/RYSER KNRDQSITYSRT }

Bane, lmesmmr lw

KNSYQS/ARNNI+K80 KNSYQSINYSYN+VZ4 frat Tl

KDSRQS/YRSDV KHHCAS/DRSRQ

Sel amen 'KNSTQS/DYSSR KNSRAQ/RNSQI

KNHHQS/QHSNR KNSGQQ/QYSRY - ’ bet ll CR

KDSRTS/AYSYK KRTYQS/RSSNT

SEER mene, v

KNSCQQ/QRSNR KNDYYS/TYSRV

EEE Emel]

KRSYQS/RYSEQ KNSYRK/KSSNI

Smee geen

KNSCASMNYSRY KNTYQS/QHSNR

Sane men

KNSSRD/QRSNI KNSYQS/KESDR

Gs aaa

KNTCQS/ECSNI KNNGQS/KYSNI

EE geseww 1v)

KSSHKS/RSNR KNDYCS/KESDR

KTSHRS/NRSRY KNTYWS/IDYSSR

KNSYHS/AYSRY KDSRGS/QNSRV

BEsemm ammew |; ¥

KDSRQS/KESDR NNSYRK/ARRNI

KNSRNQYARSRL KWSCQS/KASDK

KGSYKSITRSER KNSYGQ/KYSNQ * the underlined variants can cleave the identical target found in the Chinese Hamster GS gene,

Table III: Sequence of heterodimeric I-Crel variants cleaving a

DNA target from the Chinese Hamster GS gene

Sequence Sequence {SEQ ID NO: 109, 110, 152 to 155, 114, | (SEQID NO:12% to 133, 164 to 167, a as aE 160 to 162, 126, 127, 163 150, 175

KNCCHS/KYSNI KRSYQS/TYSRT

KRGYQS/RHRDI 30R 33R 38E 44D 68Y 70S 75Y 77Q ) 30

KRSRES/DYSYQ 30R 32G 44K B8N 30R 32C 44R 68H

Gam

KNHCQA/RYSER KRTNQQ/IRSNR

KNSROR/KYSEV KNSGQG/TYSYR

KNSNQR/IKYSEV KRDYQS/INYSYQ

ERT aeammer ow

KRSYQS/KESDR KNHHQS/RYSEY i

KNSYQS/LRNNI+K80 KNSYQS/NYSYN+V24 :

Een pe

KRSYQSIKSSNY KSTSRS/AYSDH .

KSSCQANKHNI KNGHQS/QESRR

KRSYQS/QHSNR } KTSGQSIQYSRV eww eee [%

KNRDQS/ECSNI KNSNYR/QRONR fomimmm R Ean

KSSHKS/IRSNR KNDYCS/KESDR

KNSHQT/NRSRY . KRSYES/DYSSR

EEE |, [0

KNSCQH/VERNR +K80 SNSYRK/NRSRY

Ea ee; 1 w

KDSRTS/YRSDV KKSSQS/DYSSR

Een elm

KDSRQS/KESDR NNSYRK/ARRNI

KWSCQS/KASDK

Ee eh

KGSYKS/ARSER . KNSRQR/ARGNI * the underlined variants can cleave the identical target found in the mouse GS gene.

The sequence of each I-Crel variant is defined by the mutated residues at the indicated positions. For example, the first heterodimeric variant of

Table I consists of a first monomer having T, G, V, E, N, R and K at positions 30, 33, 44, 68, 75, 77 and 80, respectively and a second monomer having R, T,N, Q,D, S, R : and T at positions 30, 32, 33, 40, 44, 70, 75, and 77 respectively. The positions are : indicated by reference to [-Crel sequence (SEQ ID NO: 1); [-Crel has N, S, Y, Q, S,

Q,R, RD, Iand E at positions 30, 32, 33, 38, 40, 44, 68, 70, 75, 77 and 80 respectively.

Each monomer (first monomer and second monomer) of the heterodimeric variant according to the present invention may also be named with a : letter code, after the eleven residues at positions 28, 30, 32, 33, 38, 40, 44, 68 and 70, 75 and 77 and the additional residues which are mutated, as indicated above. For example, KTSGQS/ENRNR + 80K or 28K30T32533G38Q40S / 44E68NT0R75N77R + 80K stands for I-Crel K28, T30, S32, G33 , S38, S40/ E44, N68, R70, N75, R77 and K80.

The heterodimeric variant as defined above may have only the amino acid substitutions as indicated above. In this case, the positions which are not . indicated are not mutated and thus correspond to the wild-type I-Crel (SEQ ID NO: 1) or I-Crel N75 scaffold (SEQ ID NO: 4) sequence, respectively. Examples of such heterodimeric I-Crel variants cleaving the GS DNA targets of figures 18 to 20 (nucleotide sequences SEQ ID NO: 5 to 60) include the variants consisting of a first and a second monomer corresponding to the following pairs of sequences: SEQ ID

NO: 61 to 84 (first monomer) and SEQ ID NO: 85 to 108, respectively (second monomer; Figure 18 and Table I}; SEQ ID NO: 109 to 123, 75, 124 to 128 (first monomer) and SEQ ID NO: 129 to 146, 99, 147 to 151, respectively (second monomer; Figure 19 and Table II); SEQ ID NO: 109, 110, 152 to 155, 114, 156 to 159, 75, 160 to 162, 126, 127, 163 (first monomer) and SEQ ID NO: 129 to 133, 164 : to 167, 137, 168 to 171, 99, 172 to 174, 149, 150 and 175, respectively (second monomer; Figure 20, Tables III and X).

Alternatively, the heterodimeric variant may consist of an I[-Crel sequence comprising the amino acid substitutions as defined above. In the latter case, the positions which are not indicated may comprise additional mutations, for example one or more additional mutations as defined above.

In particular, one or both monomers of the heterodimeric variant comprise advantageously additional substitutions that increase the cleavage activity of the variant for the GS target.

For example, the heterodimeric variants formed by a first variant having any of the sequence SEQ ID NO: 211 to 229, 242 to 244 and 271 (Tables X1 and XII) and a second variant having any of the sequence SEQ ID NO: 245 to 268 (Tables XIII and XIV) have additional substitutions that increase the cleavage activity for the GSCHO! target (SEQ ID NO: 30). oo

Preferred heterodimeric variants cleaving the GSCHOI target are presented in Table IV.

Table IV: Preferred heterodimeric I-Crel variants for the cleavage of the GSCHO1 target , 7 =F

ER ae mm [a] mmm [| =] emo * The additional mutations which improve the cleavage activity of the variant against the GSCHO.1 target are in bold

The invention encompasses I-Crel variants having at least 85 % identity, preferably at least 90 % identity, more preferably at least 95 % (96 %, 97 %, 98 %, 99 %) identity with the sequences as defined above, said variant being able to cleave a DNA target from the GS gene.

The heterodimeric variant is advantageously an obligate heterodimer variant having at least one interesting pair of mutations corresponding to residues of the first and the second monomers which make an intermolecular interaction between the two I[-Crel monomers, wherein the first mutation of said pair(s) is in the first monomer and the second mutation of said pair(s} is in the second monomer and said pair(s) of mutations prevent the formation of functional homodimers from each monomer and allow the formation of a functional! heterodimer, able to cleave the genomic DNA target from the GS gene.

To form an obligate heterodimer, the monomers have advantageously at least one of the following pairs of mutations, respectively for the first and the second monomer: a) the substitution of the glutamic acid at position 8 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at position 7 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine residues at positions 7 and 96, by an arginine, b) the substitution of the glutamic acid at position 61 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at. position 96 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine 16 residues at positions 7 and 96, by an arginine, ¢) the substitution of the leucine at position 97 with an aromatic amino acid, preferably a phenylalanine (first monomer) and the substitution of the Co phenylalanine at position 54 with a small amino acid, preferably a glycine (second monomer); the first monomer may further comprise the substitution of the phenylalanine at position 54 by a tryptophane and the second monomer may further comprise the substitution of the leucine at position 58 or lysine at position 57, by a methionine, and d) the substitution of the aspartic acid at position 137 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the arginine at position 5] with an acidic amino acid, preferably a glutamic acid (second monomer).

For example, the first monomer may have the mutation D137R and the second monomer, the mutation R51D. The obligate heterodimer meganuclease comprises advantageously, at least two pairs of mutations as defined in a), b) c) or d), above; one of the pairs of mutation is advantageously as defined in ¢) or d).

Preferably, one monomer comprises the substitution of the lysine residues at positions 7 and 96 by an acidic amino acid (aspartic acid (D) or glutamic acid (E)), preferably a glutamic acid (K7E and K96E) and the other monomer comprises the substitution of ) the glutamic acid residues at positions § and 61 by a basic amino acid (arginine (R) or lysine (K); for example, E8K and E61R). More preferably, the obligate heterodimer meganuclease, comprises three pairs of mutations as defined in a), b) and c), above.

The obligate heterodimer meganuclease consists advantageously of a first monomer (A) having at least the mutations (i) ESR, E8K or E8H, E61R, E6IK or E61H and

L97F, L97W or L97Y; (11) K7R, E8R, E61R, K96R and L97F, or (iii) K7R, ESR,

F54W, E6IR, K96R and L97F and a second monomer (B) having at least the mutations (iv) K7E or K7D, F54G or F54A and K96D or K96E; (v) K7E, F54G,

L58M and K96E, or (vi) K7E, F54G, K57M and K96E. For example, the first monomer may have the mutations K7R, E8R or E8K, E61R, K96R and L97F or K7R,

E8R or ESK, F54W, E61R, K96R and L97F and the second monomer, the mutations

K7E, F54G, L58M and K96E or K7E, F54G, K57M and K96E. The obligate heterodimer may comprise at least one NLS and/or one tag as defined above; said

NLS and/or tag may be in the first and/or the second monomer

The subject-matter of the present invention is also a single-chain chimeric meganuclease (fusion protein) derived from an I-Crel variant as defined above. The single-chain meganuclease may comprise two I-Crel monomers, two I-

Crel core domains (positions 6 to 94 of I-Crel} or a combination of both. Preferably, the two monomers /core domains or the combination of both, are connected by a peptidic linker.

The subject-matter of the present invention is also a polynucleotide fragment encoding a variant or a single-chain chimeric meganuclease as defined above; said polynucleotide may encode one monomer of a homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease.

The subject-matter of the present invention is also a recombinant vector for the expression of a variant or a single-chain meganuclease according to the invention. The recombinant vector comprises at least one polynucleotide fragment encoding a variant or a single-chain meganuclease, as defined above. In a preferred embodiment, said vector comprises two different polynucleotide fragments, each encoding one of the monomers of a heterodimeric variant.

A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or

RNA molecule which may consists of a chromosomal, non chromosomal, semi- synthetic or synthetic 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 skilled in the art and commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), para- myxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picor- navirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomega- lovirus), 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).

Preferred vectors include lentiviral vectors, and particularly self inactivacting lentiviral vectors.

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 phosphoribosy! transferase for eukaryotic cell culture; TRPI, URA3 and LEU2 for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.

Preferably said vectors are expression vectors, wherein the sequence(s) encoding the variant/single-chain meganuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said variant. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a repli- : cation origin, a promoter operatively linked to said polynucleotide, a ribosome- binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the polypeptide is expressed.

Preferably, when said variant is a heterodimer, the two polynucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-B-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), a-antitrypsin protease, human surfactant (SP)

A and B proteins, -casein and acidic whey protein genes.

According to another advantageous embodiment of said vector, it includes a targeting construct comprising sequences sharing homologies with the : region surrounding the genomic DNA cleavage site as defined above.

For instance, sald sequence sharing homologies with the regions surrounding the genomic DNA cleavage site of the variant is a fragment of the mouse

GS gene comprising positions: 2913-3112, 2971-3170, 2999-3198, 3045-3244; 4653- 4852, 6360-6559,6400-6599, 6445-6644, 7083-7282, 7105-7304, 7234-7433, 7266- 7465, 7302- 7501, 7314-7513, 7316-7515, 7423-7622, 7882-8081, 7906-8105, 7998- 8197, 8005-8204 and 8012-8211 of SEQ ID NO: 3. Alternatively, said sequence sharing homologies with the regions surrounding the genomic DNA cleavage site of the variant is a fragment of the human GS gene comprising positions: 2988-3187, 3073-3272, 3075-3274, 3081-3280, 3121-3320, 3127-3326, 4540-4739, 5405-5604, 5425-5624, 5454-5653, 5823-6022, 5936-6135, 5954-6153, 6272-6471, 6341-6540, 6986-7185, 7046-7245, 7055-7254, 7079-7278, 7089-7288, 7136-7335, 7171-7370, 7178-7377 and 7185-7384 of SEQ ID NO: 272.

Alternatively, the vector coding for an [-Crel variant/single-chain } meganuclease and the vector comprising the targeting construct are different vectors.

More preferably, the targeting DNA construct comprises: a) sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above, and b) a sequence to be introduced flanked by sequences as in a) or included in sequences as in a).

Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. Therefore, the targeting :

DNA eonstruct is preferably from 200 pb to 6000 pb, more preferably from 1000 pb to 2000 pb. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be loeated between the two arms. The sequence to be introduced may be any sequence used to alter the chromosomal DNA in some specific way including a sequence used } to repair a mutation in the GS gene, restore a functional GS gene in place of a mutated one, modify a specific sequence in the GS gene, to attenuate or activate the GS gene, to inactivate or delete the GS gene or part thereof, to introduce a mutation into a site of interest or to introduce an exogenous gene or part thereof. Such ehromosomal DNA alterations are used for genome engineering (animal models/recombinant cell lines) or genome therapy (gene correction or recovery of a functional gene). The targeting construct comprises advantageously a positive selection marker between the two homology arms and eventually a negative selection marker upstream of the first . homology arm or downstream of the second homology arm. The marker(s) allow(s) the selection of cells having inserted the sequence of interest by homologous recombination at the target site.

For example figures 18 to 20 indicate the targets from the human, mouse and Chinese Hamster GS genes, examples of variants which are able to cleave said targets and the minimal repair matrix for repairing the cleavage at each target site.

The sequence to be introduced is preferably a sequence for inactivating or deleting the GS gene or part thereof (figure 3A). Such chromosomal

DNA alterations can be used for making genetically modified cell lines wherein the endogenous GS gene is inactivated and a transgene expression cassette is eventually inserted at the GS gene locus. Such chromosomal DNA alterations can also be used for making knock-out and knock-in cell/animals wherein the GS gene is inactivated (knock-out) and eventually replaced with an exogenous gene of interest (knock-in).

Following inactivation of the endogenous GS gene, Glutamine

Synthetase may be used as a positive selection marker in further genome engineering strategies (targeted or random gene manipulation) at any locus of the genome of the

GS deficient cell/animal.

For making knock-in cells/animals, the targeting DNA construct comprises a GS gene fragment which has at least 200 bp of homologous sequence flanking the target site of the I-Crel variant for repairing the cleavage, the sequence of an exogenous gene of interest included in an expression cassette and eventually a selection marker such as the neomycin resistance gene.

For the insertion of a sequence, DNA homologies are generally located in regions directly upstream and downstream to the site of the break (sequences immediately adjacent to the break; minimal repair matrix). However, when the insertion is associated with a deletion of ORF sequences flanking the cleavage site, shared DNA homologies are located in regions upstream and downstream the region of the deletion.

Alternatively, the sequence to be introduced is a sequence which repairs a mutation in the GS gene (gene correction or recovery of a functional gene), for the purpose of genome therapy (figure 3C and 3D). For correcting the GS gene, cleavage of the gene occurs in the vicinity of the mutation, preferably, within 500 bp of the mutation (Figure 3C). The targeting construct comprises a GS gene fragment which has at least 200 bp of homologous sequence flanking the target site (minimal repair matrix) for repairing the cleavage, and includes a sequence encoding a portion of wild-type GS gene corresponding to the region of the mutation for repairing the mutation (Figure 3C). Consequently, the targeting construct for gene correction comprises or consists of the minimal repair matrix; it is preferably from 200 pb to 6000 pb, more preferably from 1000 pb to 2000 pb. Preferably, when the cleavage site of the variant overlaps with the mutation the repair matrix includes a modified cleavage site that is not cleaved by the variant which is used to induce said cleavage in the GS gene and a sequence encoding wild-type GS that does not change the open reading frame of the GS gene.

Alternatively, for restoring a functional gene (Figure 3D), cleavage ' of the gene occurs upstream of a mutation. Preferably said mutation is the first known mutation in the sequence of the gene, so that all the’ downstream mutations of the gene can be corrected simultaneously. The targeting construct comprises the exons downstream of the cleavage site fused in frame (as in the cDNA) and with a polyadenylation site to stop transcription in 3'. The sequence to be introduced (exon knock-in construct) is flanked by introns or exons sequences surrounding the cleavage site, so as to allow the transcription of the engineered gene (exon knock-in gene) into a mRNA able to code for a functional protein (Figure 3D). For example, the exon knock-in construct is flanked by sequences upstream and downstream of the cleavage site, from a minimal repair matrix as defined above.

The subject matter of the present invention is also a targeting DNA construct as defined above.

The subject-matter of the present invention is also a composition characterized in that it comprises at least one meganuclease as defined above (variant or single-chain chimeric meganuclease) and/or at least one expression vector encoding said meganuclease, as defined above.

In a preferred embodiment of said composition, it comprises a targeting DNA construct, as defined above.

Preferably, said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the meganuclease according to the invention.

The subject-matter of the present invention is further the use of a meganuclease as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for genome engineering of the GS gene for non-therapeutic purposes. The GS gene may be the endogenous GS gene at its genomic locus or a transgene that has been inserted in an animal or a cell line, for example a GS knock-in animal or cell line.

According to an advantageous embodiment of said use, it is for inducing a double-strand break in a site of interest of the GS gene comprising a genomic DNA target sequence, thereby inducing a DNA recombination event, a DNA loss or cell death.

According to the invention, said double-strand break is for: repairing a specific sequence in the GS gene, modifying a specific sequence in the GS gene, restoring a functional GS gene in place of a mutated one, attenuating or activating the

GS gene, introducing a mutation. into-a site of interest of the GS gene, introducing an exogenous gene or a part thereof, inactivating or deleting the GS gene or a part thereof, translocating a chromosomal arm, or leaving the DNA unrepaired and degraded.

Preferably it is for : (i) inactivating the GS gene by homologous recombination with an inactivation cassette (knock-out animal/cell line (figure 3A)) and eventually inserting a transgene expression cassette at the GS gene locus (knock- in animal/cell line (figure 3A) or (ii) inactivating the GS gene by non-homologous end joining (figure 3B)).

The subject-matter of the present invention is also a method for making a GS knock-out or knock-in recombinant cell, comprising at least the step of: (a) introducing into a cell, a meganuclease as defined above (I-Crel variant or single-chain derivative), so as to into-induce a double stranded cleavage at a site of interest of the GS gene comprising a DNA recognition and cleavage site for said meganuclease, simultaneously or consecutively, (b) introducing into the cell of step (a), a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a recombinant cell having repaired the site of interest by homologous recombination, (c) isolating the recombinant cell of step (b), by any appropriate means.

The subject-matter of the present invention is also a method for making a GS knock-out or knock-in animal, comprising at least the step of: (a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease as defined above, so as to induce a double stranded cleavage at a site of interest of the GS gene comprising a DNA recognition and cleavage site for said meganuclease, simultaneously or consecutively,

(b) introducing into the animal precursor cell or embryo of step (a) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of . interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a genomically modified animal precursor cell or embryo having repaired the site of interest by homologous recombination, (c) developing the genomically modified animal precursor cell or embryo of step (b) into a chimeric animal, and (d) deriving a transgenic animal from the chimeric animal of step (c). : Preferably, step (c) comprises the introduction of the genomically ‘modified precursor cell generated in step (b) into blastocysts so as to generate chimeric animals.

The targeting DNA is introduced into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest.

For making knock-out cells/animals, the DNA which repairs the site of interest comprises sequences that inactivate the GS gene.

For making knock-in cells/animals, the DNA which repairs the site of interest comprises the sequence of an exogenous gene of interest, and eventually a selection marker, such as the neomycin resistance gene.

In a preferred embodiment, said targeting DNA construct is inserted in a vector. .

Alternatively, the GS gene may be inactivated by repair of the double-strand break by non-homologous end joining (Figure 3B).

The subject-matter of the present invention is also a method for making a GS-deficient cell, comprising at least the step of: (a) introducing into a cell, a meganuclease as defined above, so as to induce a double stranded cleavage at a site of interest of the GS gene comprising a

DNA recognition and cleavage site of said meganuclease, and thereby generate genomically modified GS deficient cell having repaired the double-strands break, by non-homologous end joining, and

(b) isolating the genomically modified GS deficient cell of step(a), by any appropriate mean.

The subject-matter of the present invention is also a method for making a GS knock-out animal, comprising at least the step of: (a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease, as defined above, so as to induce a double stranded cleavage at a site of interest of the GS gene comprising a DNA recognition and cleavage site of said meganuclease, and thereby generate genomically modified precursor cell or embryo having repaired the double-strands break by non-homologous end joining, (b) developing the genomically modified animal precursor cell or embryo of step (a) into a chimeric animal, and (c) deriving a transgenic animal from a chimeric animal of step (b).

Preferably, step (b) comprises the introduction of the genomically modified precursor cell obtained in step (a), into blastocysts, so as to generate chimeric animals.

The cells which are modified may be any cells of interest. For making knock-in/transgenic mice, the cells are pluripotent precursor cells such as embryo-derived stem (ES) cells, which are well-known in the art. For making recombinant human cell lines, the cells may advantageously be PerCé (Fallaux et al.,

Hum. Gene Ther. 9, 1909-1917, 1998) or HEK293 (ATCC # CRL-1573) cells. For making mouse cell lines, the cells may advantageously be NSO, SP2/0 (BALB/c myeloma; ECACC #85110503 and #85072401), or L (ATCC #CRL-2648) cells. For making Chinese Hamster cell lines, the cells may advantageously be CHO-K1 (ATCC # CCL-61 , DG44 (Invitrogen), or CHO-S (Invitrogen) cells.

The animal is preferably a mammal, more preferably a laboratory rodent (mice, rat, guinea-pig), or a cow, pig, horse or goat.

Said meganuclease can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell.

For making recombinant cell lines expressing an heterologous protein of interest, the targeting DNA comprises a sequence encoding the product of interest (protein or RNA), and eventually a marker gene, flanked by sequences upstream and downstream the cleavage site, as defined above, so as to generate : genomically modified cells having integrated the exogenous sequence of interest in the GS gene, by homologous recombination.

The sequence of interest may be any gene coding for a certain protein/peptide of interest, included but not limited to: reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, disease causing gene products and toxins. The sequence may also encode an

RNA molecule of interest including for example a siRNA.

The expression of the exogenous sequence may be driven, either by the endogenous GS gene promoter or by a heterologous promoter, preferably a - ubiquitous or tissue specific promoter, either constitutive or inducible, as defined above. In addition, the expression of the sequence of interest may be conditional; the expression may be induced by a site-specific recombinase (Cre, FLP...).

Thus, the sequence of interest is inserted in an appropriate cassette that may comprise an heterologous promoter operatively linked to said gene of interest and one or more functional sequences including but not limited to (selectable) marker genes, recombinase recognition sites, polyadenylation signals, splice acceptor sequences, introns, tags for protein detection and enhancers.

The subject matter of the present invention is also a kit for making :

GS knock-out or knock-in cells/fanimals comprising at least a meganuclease and/or one expression vector, as defined above. Preferably, the kit further compnses a targeting DNA comprising a sequence that inactivates the GS gene flanked by sequences sharing homologies with the region of the GS gene surrounding the DNA cleavage site of said meganuclease. In addition, for making knock-in cells/animals, the kit includes also a vector comprising a sequence of interest to be introduced in the genome of said cells/animals and eventually a selectable marker gene, as defined above. ~The subject-matter of the present invention is also the use of at least one meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing a pathological condition caused by a mutation in the GS gene as defined above, in an individual in need thereof.

Preferably said pathological condition is inherited systemic deficiency of glutamine.

The use of the meganuclease may comprise at least the step of (a) . inducing in somatic tissue(s) of the donor/ individual a double stranded cleavage at a site of interest. of the GS gene comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into said somatic tissue(s) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the GS gene upon recombination between the targeting

DNA and the chromosomal DNA, as defined above. The targeting DNA is introduced into the somatic tissues(s) under conditions appropriate for introduction of the . targeting DNA into the site of interest.

According to the present invention, said double-stranded cleavage may be induced, ex vivo by introduction of said meganuclease into somatic cells from the diseased individual and then transplantation of the modified cells back into the diseased individual.

The subject-matter of the present invention is also a method for preventing, improving or curing a pathological condition caused by a mutation in the

GS gene, in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means. The meganuclease can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into mouse cells, by any convenient means well-known to those in the art, which are appropriate for the particular cell type, alone or in association with either at least an appropriate vehicle or carrier and/or with the targeting DNA.

According to an advantageous embodiment of the uses according to the invention, the meganuclease (polypeptide) is associated with: - liposomes, polyethyleneimine (PEI); in such a case said association is administered and therefore introduced into somatic target cells. - membrane translocating peptides (Bonetta, The Scientist, 2002, 16, 38; Ford ef al., Gene Ther., 2001, 8, 1-4 ; Wadia and Dowdy, Curr. Opin. Biotechnol. 2002, 13, 52-56); in such a case, the sequence of the variant/single-chain meganuclease is fused with the sequence of a membrane translocating peptide (fusion protein).

According to another advantageous embodiment of the uses according to the invention, the meganuclease (polynucleotide encoding said meganuclease) and/or the targeting DNA is inserted in a vector. Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods {e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”).

Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.

Once in a cell, the meganuclease and if present, the vector comprising targeting DNA and/or nucleic acid encoding a meganuclease are imported or translocated by the cell from the cytoplasm to the site of action in the nucleus.

For purposes of therapy, the meganucleases and a pharmaceutically acceptable excipient are administered in a therapeutically effective amount. Such a combination is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality.

In one embodiment of the uses according to the present invention, the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response. A variety of methods for ameliorating or eliminating delete- rious immunological reactions of this sort can be used in accordance with the inven- tion. In a preferred embodiment, the meganuclease is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate meganucleases to polyethylene glycol ("PEG") or polypropylene glycol ("PPG") (preferably of 500 to 20,000 daltons average molecular weight (MW)). Conjugation with PEG or PPG, as described by Davis et al (US 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble endonuclease conju- gates with anti-viral activity. Similar methods also using a polyethylene--poly- propylene glycol copolymer are described in Saifer ef al. (US 5,006,333). : The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector. : :

The invention also concerns a non-human transgenic animal or a transgenic plant, characterized in that all or a part of their cells are modified by a polynucleotide or a vector as defined above.

As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or an eukaryotic cell, such as an animal, plant or yeast cell.

The subject-matter of the present invention is also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example, further rounds of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel meganucleases.

The different uses of the meganuclease and the methods of using said meganuclease according to the present invention include the use of the I-Crel variant, the single-chain chimeric meganuclease derived from said variant, the poly- nucleotide(s), vector, cell, transgenic plant or non-human transgenic mammal encoding said variant or single-chain chimeric meganuclease, as defined above.

The [-Crel variant according to the invention may be obtained by a method for engineering [-Crel variants able to cleave a genomic DNA target sequence from the GS gene, comprising at least the steps of: (a) constructing a first series of I-Crel variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 28 to 40 of I-Crel, (b) constructing a second series of I-Crel variants having at least: one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of I-Crel, (¢) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet at positions -10 to -8 of the I-Crel site has been replaced. with the nucleotide triplet which is present at positions -10 to -8 of said genomic target and (ii) the nucleotide triplet at positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions -10 to -8 of said genomic target,

: (d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet at positions -5 to -3 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions -5 to -3 of said genomic target and (ii) the nucleotide triplet at positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions -5 to -3 of said genomic target,

(e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet at positions +8 to +10 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions +8 to +10 of said genomic target and (ii) the nucleotide triplet at positions -10 to -8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions +8 to +10 of said genomic target,

(f) selecting and/or screening the variants from the second series of

* step (b) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet at positions +3 to +5 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions +3 to +5 of said genomic target and (ii) the nucleotide : triplet at positions -5 to -3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions +3 to +5 of said genomic target, (g) combining in a single variant, the mutation(s) at positions 28 to 40 and 44 to 77 of two variants from step (c) and step (d), to obtain a novel homodimeric 1-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet at positions -10 to -8 is identical to the nucleotide triplet which is present at positions -10 to -8 of said genomic target, (ii) the nucleotide triplet at positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions -10 to -8 of said genomic target, (iii) the nucleotide triplet at positions -5 to -3 is identical to the nucleotide triplet which is present at positions -5 to -3 of said genomic target and (iv) the nucleotide triplet at positions +3 to +5 is identi- cal to the reverse complementary sequence of the nucleotide triplet which is present at positions -5 to -3 of said genomic target, and/or. . (h) combining in a single variant, the mutation(s) at positions 28 to 40 and 44 to 77 of two variants from step (e} and step (f), to obtain a novel homodimeric [-Crel variant which cleaves a sequence wherein (1) the nucleotide triplet at positions +3 to +5 is identical to the nucleotide triplet which is present at positions +3 to +5 of said genomic target, (ii) the nucleotide triplet at positions -5 to -3 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions +3 to +5 of said genomic target, (111) the nucleotide triplet at positions +8 to +10 of the I-Crel site has been replaced with the nucleotide triplet : which is present at positions +8 to +10 of said genomic target and (iv) the nucleotide triplet at positions -10 to -8 is identical to the reverse complementary sequence of the - nucleotide triplet at positions +8 to +10 of said genomic target, (i) combining the variants obtained in steps {(g) and (h} to form heterodimers, and (5) selecting and/or screening the heterodimers from step (1) which are able to cleave said genomic DNA target from the GS gene.

One of the step(s) (c), (d), (e) or (f) may be omitted. For example, if step (c) is omitted, step (d) is performed with a mutant [-Crel site wherein both nucleotide triplets at positions -10 to -8 and -5 to -3 have been replaced with the nucleotide triplets which are present at positions -10 to -8 and -5 to -3, respectively of said genomic target, and the nucleotide triplets at positions +3.to +5 and +8 to +10 have been replaced with the reverse complementary sequence of the nucleotide triplets ‘which are present at positions -5 to -3 and -10 to -8, respectively of said genomic target.

The (intramolecular) combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques.

The (intermolecular) combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), s0 as to allow the formation of heterodimers. For example, host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the varant(s), so that heterodimers are formed in the host cells, as described previously in the International

PCT Application WO 2006/097854 and Amould ef al., J. Mol. Biol., 2006, 355, 443- 458.

The selection and/or screening in steps (c), (d), (e), (f) and/or (j) may be performed by using a cleavage assay in vifro or in vivo, as described in the

International PCT Application WO 2004/067736, Amould ef al., J. Mol. Biol., 2006, 355, 443-458, Epinat et al., Nucleic Acids Res., 2003, 31, 2952-2962 and Chames ef al., Nucleic Acids Res., 2005, 33, 178.

According to another advantageous embodiment of said method, steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination- mediated repair of said DNA double-strand break.

Furthermore, the homodimeric combined variants obtained in step (g) or (h) are advantageously submitted to a selection/screening step to identify those which are able to cleave a pseudo-palindromic sequence wherein at least the nucleotides at positions -11 to -3 (combined variant of step (g)) or +3 to +11 (combined variant of step (h)) are identical to the nucleotides which are present at positions -11 to -3 (combined variant of step (g)) or +3 to +11 (combined variant of step (h)) of said genomic target, and the nucleotides at positions +3 to +11 (combined variant of step (g)) or -11 to -3 (combined variant of step (h)) are identical to the reverse complementary sequence of the nucleotides which are present at positions -11 to -3 (combined variant of step (g)}) or +3 to +11 (combined variant of step (h)) of said genomic target.

Preferably, the set of combined variants of step (g) or step (h} (or both sets) undergoes an additional selection/screening step to identify the variants which are able to cleave a pseudo-palindromic sequence wherein : (i) the nucleotides at positions -2 to +2 (four central bases) are identical to the nucleotides which are present at positions -2 to +2 of said genomic target, (ii} the nucleotides at positions - 11 to -3 (combined variant of step g}) or +3 to +11 (combined variant of step (h)} are identical to the nucleotides which are present at positions -11 to -3 (combined variant of step (g)} or +3 to +11 (combined variant of step h)} of said genomic target, and (iii) the nucleotides at positions +3 to +11 (combined variant of step (g)) or -11 to -3 (combined variant of step (h)) are identical to the reverse complementary sequence of the nucleotides which are present at positions -11 to -3 (combined variant of step (g)) or +3 to +11 (combined variant of step (h)) of said genomic target. This additional screening step increases the probability of isolating heterodimers which are able to cleave the genomic target of interest (step (j)).

Steps (a), (b), (g), (h} and (i) may further comprise the introduction of additional mutations at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target, at positions which improve the binding and/or cleavage properties of the variants, or at positions which either prevent or impair the formation of functional homodimers or favor the formation of the heterodimer, as defined above.

The additional mutations may be introduced by site-directed mutagenesis and/or random mutagenesis on a variant or on a pool of variants, according to standard mutagenesis methods which are well-known in the art, for example by using PCR.

In particular, random mutations may be introduced on the whole variant or in a part of the variant, in particular the C-terminal half of the variant (positions 80 to 163) to improve the binding and/or cleavage properties of the variants towards the DNA target from the gene of interest. Site-directed mutagenesis at positions which improve the binding and/or cleavage properties of the variants, for example at positions 19, 54, 80, 87, 105 and /or 132, may also be combined with random-mutagenesis. The mutagenesis may be performed by generating random/site- directed mutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art. Site-directed mutagenesis may be advantageously performed by amplifying overlapping fragments comprising the

: mutated position(s), as defined above, according to well-known overlapping PCR techniques. In addition, multiple site-directed mutagenesis, may advantageously be : ,performed on a variant or on a pool of variants,

Preferably, the mutagenesis is performed on one monomer of the heterodimer formed in step (i) or obtained in step (j), advantageously on a pool of monomers, preferably on both monomers of the heterodimer of step (i) or (j).

Preferably, at least two rounds of selection/screening are performed according to the process illustrated by figure 4 of Amould ef al., J. Mol. Biol., 2007, 371, 49-65. In the first round, one of the monomers of the heterodimer is mutagenised (monomer Y in figure 4), co-expressed with the other monomer (monomer X in figure 4) to form heterodimers, and the improved monomers Y" are selected against the © target from the gene of interest. In the second round, the other monomer (monomer X) is mutagenised, co-expressed with the improved monomers Y" to form heterodimers, and selected against the target from the gene of interest to obtain meganucleases (X*

YY") with improved activity. The mutagenesis may be random-mutagenesis or site- directed mutagenesis on a monomer or on a pool of monomers, as indicated above.

Both types of mutagenesis are advantageously combined. Additional rounds of selection/screening on one or both monomers may be performed to improve the . cleavage activity of the variant.

The cleavage activity of the improved meganuclease obtainable by the method according to the present invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector, by comparison with that of the initial meganuclease. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic

DNA target sequence which is cleaved by the initial meganuclease, within the intervening sequence, cloned in a yeast or a mammalian expression vector. Expression of the meganuclease results in cleavage of the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene (LacZ, for example), whose expression can be monitored by appropriate assay. A stronger signal is observed with the improved meganuclease, as compared to the initial meganuclease. Altematively, the activity of the improved meganuclease towards its genomic DNA target can be compared to that of I-Crel towards the I-Crel site, at the same genomic locus, using a chromosomal assay in mammalian cells (Amould ef ai., J. Mol. Biol., 2007, 371, 49-65).

The subject matter of the present invention is also an I-Crel variant having mutations at positions 28 to 40 and/or 44 to 77 of I-Crel that is useful for engineering the variants able to cleave a DNA target from the GS gene, according to the present invention. In particular, the invention encompasses the I-Crel variants as defined in step (c) to (f) of the method for engineering I-Crel variants, as defined above, including the variants at positions 28, 30, 32, 33, 38 and 40, or 44, 68, 70, 75 and 77 presented in Tables V and VII. The invention encompasses also the I-Crel variants as defined in step (g) and (h) of the method for engineering I-Crel variants, as defined above including the combined variants of Table V to VIII.

Single-chain chimeric meganucleases able to cleave a DNA target from the gene of interest are derived from the variants according to the invention by methods well-known in the art (Epinat et af., Nucleic Acids Res., 2003, 31, 2952-62;

Chevalier er al., Mol. Cell, 2002, 10, 895-905; Steuer e! al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention.

The polynucleotide sequence(s) encoding the variant as defined in the present invention may be prepared by any method known by the man skilled in the art, For example, they are amplified from a ¢cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.

The recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.

The I-Crel variant or single-chain derivative as defined in the present invention are produced by expressing the polypeptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed (in the case of the variant only) in a host cell or a transgenic animal/plant modified by one expression vector or two expression vectors (in the case of the variant only), under conditions suitable for the expression or co-expression of the polypeptide(s), and the variant or single-chain derivative is recovered from the host cell culture or from the transgenic animal/plant.

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 I-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 addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the I-Crel meganuclease variants and their uses according to the invention, as well as to the appended drawings in which: - Figure 1: Modular structure of homing endonucleases and the combinatorial approach for custom meganucleases design. A. Tridimensional structure of the I-Crel homing endonuclease bound to its DNA target. The catalytic core is surrounded by two afpappae folds forming a saddle-shaped interaction interface above the DNA major groove. B. Different 1-Crel variants binding different sequences derived from the I-Crel target sequence (top right and bottom left) to obtain heterodimers or single chain fusion molecules cleaving non palindromic chimeric targets (bottom right). C." The identification of smaller independent subunit, i.e., subunit within a single monomer or afpaffa fold (top right and bottom left) would allow for the design of novel chimeric molecules (bottom right), by combination of mutations within a same monomer. Such molecules would cleave palindromic chimeric targets (bottom right). D. The combination of the two former steps would allow a larger combinatorial approach, involving four different subdomains. In a first step, couples of novel meganucleases could be combined in new molecules (“half- meganucleases”) cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such “half-meganuclease” can result in a heterodimeric species cleaving the target of interest. Thus, the identification of a small number of new cleavers for each subdomain would allow for the design of a very large number of novel endonucleases. - Figure 2; Glutamine Synthetase coding sequence. A. The mouse

Glutamine Synthetase gene (accession number NC000067.5). Exons are indicated as grey boxes. The GSCHO1 target is indicated with its sequence and position. B. The

Criteculus griseus Glutamine Synthetase mRNA (accession number X03495). The

ORF is indicated as a grey box. The GSCHOI genomic target site is indicated with its sequence and its position relative to the Glutamine Synthetase mRNA sequence. - Figure 3: Strategies for the utilization of a meganuclease cleaving the Glutamine Synthetase (GS) gene. A. Gene insertion and/or gene inactivation.

Upon cleavage by a meganuclease and recombination with a repair matrix containing a gene of interest (gene insertion) or an inactivation cassette (gene inactivation), flanked by sequences sharing homology with the sequences surrounding the cleavage site, gene insertion or gene inactivation occurs. B. Gene inactivation by Non-

Homologous End-Joining. Upon cleavage by a meganuclease, the DNA ends are degraded and rejoined by Non-Homologous-End-Joining (NHEI), and gene inactivation occurs. C. Gene Correction. A mutation occurs within the GS gene. Upon cleavage by a meganuclease and recombination with a repair matrix the deleterious mutation is corrected. D. Exonic sequences knock-in. A mutation occurs within the

GS gene. The mutated mRNA transcript is featured below the gene. In the repair matrix, exons located downstream of the cleavage site are fused in-frame (as in a cDNA), with a polyadenylation site to stop transcription at the 3’ end. Intronic and exonic sequences can be used as homologous regions. A knock-in of exonic sequences results in an engineered gene, transcribed into a mRNA able to code for a functional protein, - Figure 4: The GSCHOI1 target sequences and its derivatives. 10GCC_P, 10GGA_P, 5AGG _P and STTC_P are close derivatives cleaved by previously obtained [-Crel variants. They differ from C1221 by the boxed motives.

C1221, 10GCC_P, 10GGA_P, SAGG_P and 5TTC_P were first described as 24 bp sequences, but structural data suggest that only the 22 bp are relevant for protein/DNA interaction. However, positions +12 are indicated in parenthesis. GSCHOI is the ’ DNA sequence located in the mouse and Criteculus griseus Glutamine Synthetase gene. In the GSCHO!.2 target, the GTGA sequence in the middle of the target is replaced with’ GTAC, the bases found in C1221. GSCHOL1.3 is the palindromic : 15 sequence derived from the left part of GSCHO1.2, and GSCHOI 4 is the palindromic sequence derived from the right part of GSCHOI1.2. As shown in the Figure, the boxed motives from 10GCC_P, 10GGA_P, SAGG_P and 5TTC_P are found in the

GSCHOI series of targets. 3 Figure 5: pCLS1055 plasmid map. - Figure 6: pCLS0542 plasmid map. - Figure 7: Cleavage of GSCHOQ1.3 target by combinatorial variants.

The figure displays an example of screening of I-Crel combinatorial variants with the

GSCHOL1.3 target. On the filter, the sequence of the positive variant at position H2 is

KRSRES/DYSYQ (according to the nomenclature of Table VI). H10, H11, H12 are negative and positive controls of different strength. - Figure 8: pCLS1107 plasmid map. - Figure 9: Cleavage of GSCHO1.4 target by combinatorial variants.

The figure displays an example of screening of [-Crel combinatorial variants with the

GSCHOI1 4 target. HI0, H1l and HI2 are negative and positive controls of different strength. On the filter, the sequence of the positive variants at positions D4, F3 and F9 are KRDYQS/RHRDI, KRGYQS/KARDI and KRDYQS/RNRDI, respectively (according to the nomenclature of Table VII).

- Figure 10: Cleavage of the GSCHO1.2 and GSCHOI target sequences by heterodimeric combinatorial variants. A. Example of screening of combinations of I-Crel variants against the GSCHO1.2 target. B. Screening of the same combinations of 1-Crel variants against the GSCHOI target.

All heterodimers tested resulted in cleavage of the GSCHO.2 target.

The heterodimers displaying the strongest signal with the GSCHOI target are observed at positions D3, D7, D9 and E2, corresponding to yeast co-expressing the

GSCHOI1.3 variant KRSRES/DYSYQ with the GSCHOl.4 variants

KRGYQS/KHRDI, KRGYQS/KNRDI, KRCYQS/RHRDI or KRGYQS/RHRDI, respectively. E10, Ell and E12 are negative and positive controls of different strength. - Figure 11: Cleavage of the GSCHOI target. Example of screening against the GSCHOI target of I-Crel refined variants obtained by random mutagenesis of variants cleaving GSCHO1.3 (example 5) and co-expressed with a variant cutting GSCHO1.4 (KRGYQS/KNRDI according to Table VIII).

Each cluster contains 6 spots: In the 4 left spots, the yeast strain containing the

GSCHOI target and the GSCHO1.4 variant are mated with 4 different clones from the library (except for H10, H11 and HI2: negative and positive controls of different strength). The top right spot is the GSCHOI.4 variant / GSCHOI target strain mated with one of the initial GSCHOI.3 variants KRSRES/DYSYQ (according to the nomenclature of Table VI); the lower right spot is an internal control. On the filter, the sequence of the positive variants at positions C11, E12 and Fl are 30R,33R,38E,44D,66H,68Y,708,75Y,77Q,132V; 7E,19A,30R,33R,38E,44D,68Y, 708,75Y,77Q,120A, and 30R,33R,38E,44D,68Y,708,75Y,77Q,87L, respectively. - Figure 12: Cleavage of the GSCHOI! target. Example of screen against the GSCHOI target of the libraries constructed in example 6 by site-directed mutagenesis of initial variants cleaving the GSCHOQ1.3 target and co-expressed with a variant cutting GSCHO1.4 (KRGYQS/KNRDI according to Table VIII).

Each cluster contains 6 spots: For each spot, the yeast strain containing the GSCHO1 target and the GSCHOI1.4 variant is mated with; 2 different clones from the library containing the E80K ‘substitution (left spots) 2 different clones from the F87L library (middle spots), or KRSRES/DYSYQ, a variant cleaving GSCHOI.3 described in example 3 (upper right spot). The lower right spot is an internal control. H10, H11 and

H12 are negative and positive controls of different strength. The sequence of the positive variants at positions B7, and G6 are 30R,33R,38E,44D,68Y,708,75Y,77Q,80K, and 30R,33R,38E,44D,68Y,70S,75Y,77Q, 87L, respectively. : - Figure 13: Cleavage of the GSCHOI target. Example of screen against the GSCHOIl target of I-Crel refined variants obtained by random mutagenesis of variants cleaving GSCHOI1.4 (example 7) and co-expressed with a : variant cutting GSCHO1.3 (KRSRES/DYSYQ according to Table VI).

Each cluster contains 6 spots: In the 4 left spots, the yeast strain containing the

GSCHOI target and the GSCHO1.3 variant are mated with 4 different clones from the library (except for H10, HI11 and H12: negative and positive controls of different strength). The top right spot is the GSCHO1.3 variant / GSCHOI target strain mated with one of the initial GSCHO1.4 variants KRGYQS/KYSNI (according to the nomenclature of Table VIII); the lower right spot is an internal control. On the filter, the sequence of the positive variants at positions E6, DS and H3 are 30R,32G,44R,68H,132V,154G; 30R,33H,68A,77R, and 2S,30R,33H,68A,77R, respectively. - Figure 14: Cleavage of the GSCHOI1 target. Example of screen against the GSCHOI target of the libraries constructed in example 8 by site-directed mutagenesis of initial variants cleaving the GSCHOQI .4 target and co-expressed with a variant cutting GSCHO1.3 (KRSRES/DYSYQ according to Table VI).

Each cluster contains 6 spots: For each spot, the yeast strain containing the GSCHO1 target and the GSCHOI1.3 variant is mated with; 2 different clones from the library containing the G19S substitution (top 2 spots) 2 different clones from the F54L library (bottom 2 spots), or KRGYQS/KYSNI, a variant cleaving GSCHOI1.4 described in example 4 (upper right spot). The lower right spot is an internal control. H10, H11 and

H12 are negative and positive controls of different strength. The sequence of the positive variants at positions B2, Fl, and H2 are 30R,32G,44R,54L,68H; 198,30R,32G,44K,45M,68H and 19S,30R,33H,68A,77R, respectively. - Figure 15: pCLS1058 plasmid map. - Figure 16: pCLS1768 plasmid map.

- Figure 17: GSCHOL target cleavage in CHO cells.

Extrachromosomal cleavage efficiency of the GSCHOI1 target sequence in mammalian cells was compared for twelve heterodimeric combinations. The sequences of the variants tested are described in table XV. The negative control pCLS1768 is an empty expression vector. - Figure 18 represents meganuclease target sequences found in the human GS gene and examples of [-Crel variants which are able to cleave said DNA targets; at least one example of variant (heterodimer formed by a first and a second I-

Crel variant monomer) is presented for each target. The exons closest to the target sequences, and the exon junctions are indicated (columns 1 and 2), the sequence of the

DNA target is presented (column 3), with its sequence identification number (column 4) and the position of its first nucleotide by reference to human GS gene sequence (9782 bp; accession number NC 000001.9; column 5). The minimum repair matrix for repairing the cleavage at the target site is indicated by its first nucleotide (start, column 10) and last nucleotide (end, column 11). The sequence of each I-Crel variant is defined by the mutated residues at the indicated positions (columns 6 and 8) and the corresponding sequence identification number (columns 7 and 9). For example, the first heterodimeric variant of figure 18 consists of a first monomer having T, G, V, E,

N, R and K at positions 30, 33, 44, 68, 75, 77 and 80, respectively and a second monomer having R, T,N, Q,D, S, Rand T at positions 30, 32, 33, 40, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-Crel sequence (SEQ

IDNO: 1); I-Crel has N, S, Y, S, Q,R, R, D, I and E, at positions 30, 32, 33, 40, 44, 68, 70, 75, 77 and 80, respectively. - Figure 19 represents meganuclease target sequences found in the mouse GS gene and examples of I-Crel variants which are able to cleave said DNA targets; at least one example of variant (heterodimer formed by a first and a second I-

Crel variant monomer) is presented for each target. The exons closest to the target sequences, and the exon junctions are indicated (columns 1 and 2), the sequence of the

DNA target is presented (column 3), with its sequence identification number (column 4) and the position of its first nucleotide by reference to mouse GS gene sequence (SEQ ID NO: 3; column 5). The minimum repair matrix for repairing the cleavage at the target site is indicated by its first nucleotide (start, column 10) and last nucleotide

(end, column 11). The sequence of each I-Crel variant is defined by the mutated residues at the indicated positions (columns 6 and 8) and the corresponding sequence identification number (columns 7 and 9). For example, the first heterodimeric variant of figure 19 consists of a first monomer having C, C, H, K, Y, S and N at positions 32, 33, 38, 44, 68, 70 and 75, respectively and a second monomer having R, T, Y, S, R and T at positions 30, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-Crel sequence (SEQ ID NO: 1); I-Crel has N, S, Y, Q, Q,R,R, D, I and E, at positions 30, 32, 33, 38, 44, 68, 70, 75, 77 and 80 respectively. - Figure 20 represents meganuclease target sequences found in the

Chinese Hamster (Criteculus griseus.) GS gene and examples of I-Crel variants which are able to cleave said DNA targets; at least one example of variant (heterodimer formed by a first and a second I-Crel variant monomer) is presented for each target.

The exons closest to the target sequences, are indicated (columnl), the SOqUETS of the

DNA target is presented (column 2), with its sequence identification number (column ~~ 3) and the position of its first nucleotide by reference to Chinese Hamster GS mRNA sequence (GenBank X03495; column 4). The sequence of each I-Crel variant is defined by the mutated residues at the indicated positions (columns 5 and 7) and the corresponding sequence identification number (columns 6 and 8). For example, the first heterodimeric variant of figure 20 consists of a first monomer having C, C, H, K,

Y, S and N at positions 32, 33, 38, 44, 68, 70 and 75, respectively and a second - monomer having R, T, Y, §, Rand T at positions 30, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-Crel sequence (SEQ ID

NO: 1);I-Crel has N, §, Y, Q, Q, R, R, D, I and E, at positions 30, 32, 33, 38, 44, 68, 70, 75, 77 and 80 respectively.

Example 1: Strategy for engineering novel meganucleases cleaving a target from the Glutamine Synthetase (GS) gene

GSCHOQ1 is a 22 bp (non-palindromic) target located in the coding sequence of both the mouse and the Criteculus griseus (Chinese Hamster) Glutamine

Synthetase gene. The target sequence corresponds to positions 3060-3083 of the mouse Glutamine Synthetase gene (accession number NC000067.5; Figure 2A) and positions 204 to 227 of the Criteculus griseus Glutamine Synthetase (GS) cDNA (accession number X03495; Figure 2B).

: The GSCHOI sequence is partly a patchwork of the 10GCC_P, 10GGA_P, 5AGG_P and 5_TTC_P targets (Figure 4) which are cleaved by previously identified meganucleases, obtained as described in International PCT Applications

WO 2006/097784 and WO 2006/097853; Amould et al., J. Mol. Biol., 2006, 355, 443-458; Smith er al., Nucleic Acids Res., 2006. Thus, GSCHO1 could be cleaved by combiriatorial variants resulting from these previously identified meganucleases.

The 10GCC_P, 10GGA_P, SAGG_P and 5_TTC_P target sequences are 24 bp derivatives of C1221, a palindromic sequence cleaved by I-Crel (Amould et al., precited). However, the structure of I-Crel bound to its DNA target suggests that the two external base pairs of these targets (positions -12 and 12) have no impact on binding and cleavage (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774; Chevalier et al., J. Mol.

Biol, 2003, 329, 253-269), and in this study, only positions -11 to 11 were © considered. Consequently, the GSCHOI1 series of targets were defined as 22 bp sequences instead of 24 bp. GSCHOI differs from C1221 in the 4 bp central region.

According to the structure of the I-Crel protein bound to its target, there is no contact between the 4 central base pairs (positions -2 to 2) and the I-Crel protein (Chevalier et al, Nat. Struct. Biol., 2001, 8, 312-316; Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774; Chevalier et al., J. Mol. Biol., 2003, 329, 253-269). Thus, the bases at these positions should not impact the binding efficiency. However, they could : affect cleavage, which results from two nicks at the edge of this region. Thus, the gtga sequence in -2 to 2 was first substituted with the gtac sequence from C1221, resulting in target GSCHO1.2 (Figure 4). Then, two palindromic targets, GSCHOL.3 and

GSCHOI1.4, were derived from GSCHOI1.2 (Figure 4). Since GSCHOL.3 and

GSCHOL.4 are palindromic, they should be cleaved by homodimeric proteins. Thus, proteins able to cleave the GSCHOI.3 and GSCHO1.4 sequences as homodimers were first designed (examples 2 and 3) and then co-expressed to obtain heterodimers cleaving GSCHOQO | (example 4). Heterodimers cleaving the GSCHO!.2 and GSCHO! targets could be identified. In order to improve cleavage activity for the GSCHOI target, a series of variants cleaving GSCHO1.3 and GSCHO1.4 was chosen, and then refined. The chosen variants were subjected to random or site-directed mutagenesis, and used to form novel heterodimers that were screened against the GSCHO! target

(examples 5, 6, 7 and 8). Heterodimers could be identified with an improved cleavage activity for the GSCHOI target. Chosen heterodimers were subsequently cloned into mammalian expression vectors and screened against the GSCHO1 target in CHO cells (example 9). Strong cleavage activity of the GSCHOI target could be observed for these heterodimers in mammalian cells.

Example 2: Identification of meganucleases cleaving GSCHO1.3

This example shows that [-Crel variants can cut the GSCHO1.3

DNA target sequence derived from the left part of the GSCHOL.2 target in a palindromic form (Figure 4). Target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix _P (For example, target GSCHO1.3 will be noted tgccccagggt P). :

GSCHOL1.3 is similar to 10GCC_P at positions *1, £2, £6, £8, £9, and £10 and to SAGG_P at positions +1, +2, +3, +4, +5 and +6. It was hypothesized. that positions +7 and +11 would have little effect on the binding and cleavage activity.

Variants able to cleave the 10GCC_P target were obtained by mutagenesis of I-Crel

N75 or D75, at positions 28, 30, 32, 33, 38, 40 and 70, as described previously in

Smith er al. Nucleic Acids Res., 2006, 34, e149; International PCT Applications WO 2007/060495 and WO 2007/049156. Variants able to cleave SAGG_P were obtained by mutagenesis on [-Crel N75 at positions 24, 44, 68, 70, 75 and 77 as described in

Amould et al., J. Mol. Biol., 2006, 355, 443-458; Smith er al. Nucleic Acids Res., 2006, 34, e149; Intemational PCT Applications WO 2006/097784, WO 2006/097853,

WO 2007/060495 and WO 2007/049156.

Both sets of proteins are mutated at position 70. However, the existence of two separable functional subdomains was hypothesized. This implies that this position has little impact on the specificity at bases 10 to 8 of the target.

Mutations at positions 24 found in variants cleaving the SAGG_P target will be lost during the combinatorial process. But it was hypothesized that this will have little impact on the capacity of the combined variants to cleave the GSCHOI.3 target.

Therefore, to check whether combined variants could cleave the GSCHO1.3 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving SAGG_P were combined with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10GCC_P. :

A) Material and Methods a) Construction of target vector

The target was cloned as follows: an oligonucleotide corresponding to the GSCHOL.3 target sequence flanked by gateway cloning sequences was ordered from PROLIGO: 5° tggcatacaagtttctgecccagggtacectggggcageaategtctgtea 3° (SEQ ID

NO: 183). Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into the yeast reporter vector (pCLS1055, Figure 5). Yeast reporter vector was transformed into Saccharomyces cerevisiae strain FYBL2-7B (MAT a, ura3 A851, trpl A63, leu2 Al, lys2 A202), resulting in a reporter strain. b) Mating of meganuclease expressing clones and screening in yeast

I-Crel variants cleaving 10GCC_P or 5SAGG_P were previously identified, as described in Smith ef al. Nucleic Acids Res., 2006, 34, el49;

International PCT Applications WO 2007/060495 and WO 2007/049156, and Amould et al, J. Mol. Biol, 2006, 355, 443-458; International PCT Applications WO 2006/097784 and WO 2006/097853, respectively for the 10GCC_P and SAGG_P targets. In order to generate I-Crel derived coding sequences containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5” end (aa positions 1-43) or the 3’ end (positions 39-167) of the I-Crel coding sequence. For both the 5’ and 3’ end, PCR amplification is carried out using primers (GallOF 5’-gcaactttagtgctgacacatacagg-3> (SEQ ID NO: 186) or GallOR 5’- acaaccttgattggagacttgace-3’(SEQ ID NO: 187)) specific to the vector (pCLS0542,

Figure 6) and primers (assF 5’-ctannnttgaccttt-3’ (SEQ ID NO: 188) or assR 5’- aaaggtcaannntag-3'(SEQ ID NO: 189)), where nnn codes for residue 40, specific to the I-Crel coding sequence for amino acids 39-43. The PCR fragments resulting from the amplification reaction realized with the same primers and with the same coding sequence for residue 40 were pooled. Then, each pool of PCR fragments resulting from the reaction with primers GallOF and assR or assF and GallOR was mixed in an : equimolar ratio. Finally, approximately 25 ng of each final pool of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS0542, Figure 6)

linearized by digestion with Ncol and Eagl were used to transform the yeast

Saccharomyces cerevisiae strain FYC2-6A (MATa, tpl A63, leu2Al, his3A200) using a high efficiency LiAc transformation protocol (Gietz and Woods, Methods :

Enzymol., 2002, 350, 87-96). An intact coding sequence containing both groups of mutations is generated by in vivo homologous recombination in yeast. ¢) Mating of meganuclease expressing clones and screening in yeast

Screening was performed as described previously (Amould et al., J.

Mol. Biol, 2006, 355, 443-458). Mating was performed using a colony gridder (Qpixll, GENETIX). Variants were gridded on nylon filters covering YPD plates, using a low gridding density (4-6 spots/em’®). A second gridding process was performed on the same filters to spot a second layer consisting of the reporter- harboring yeast strain. Membranes were placed on solid agar YPD rich medium, and incubated at 30 °C for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (2 %) as a carbon source, and incubated for five days at 37 °C, to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02 % X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1 % SDS, 6% dimethyl formamide (DMF), 7 mM B-mercaptoethanol, 1% agarose, and incubated at 37°C, to monitor f-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software. d) Sequencing of variants

To recover the variant expression plasmids, yeast DNA was extracted using standard protocols and used to transform E. coli. Sequencing of variant ORFs was then performed on the plasmids by MILLEGEN SA. Alternatively,

ORFs were amplified from yeast DNA by PCR (Akada et al., Biotechniques, 2000, 28, 668-670), and sequencing was performed directly on the PCR product by

MILLEGEN SA.

B) Results 1-Crel combinatorial variants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving SAGG_P with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10GCC_P on the I-Crel scaffold, resulting in a library of complexity 2303. Examples of combinatorial variants are displayed in Table V. This library was transformed into yeast and 4608 clones (2 times the diversity) were screened for cleavage against the GSCHO1.3 DNA target (tgccccagggt P). Two positive clones were found (one strong cutter and one weak cutter), which after sequencing turned out to correspond to 2 different novel endonuclease variants (Table VI). Examples of positives are shown in Figure 7. These two variants display non parental combinations at positions 28, 30, 32, 33, 38, 40 or 44, 68, 70, 75, 77. Such combinations likely result from PCR artifacts during the combinatorial process. Alternatively, the variants may be I-Crel combined variants resulting from micro-recombination between two original variants during in vivo homologous recombination in yeast.

Table V: Panel of variants* theoretically present in the combinatorial library

Amino acids at . positions 44, 68, . 706. Amino aclds at positions 28, 30,32, 33, 38 and 40 75 and (ex: KHS5QS stands for K28, H30, 532, 533, Q38 and $40} . ki ih | see

ARNNI stands for

Ad4, R68,

N75 and 7

Aw [0 — rr 1 1 rr rr 1 rr 7] 00] ( Amer | rT rr "1 1 ssw [ [rr — { -r {1 rr or] oarsyy [| t+ ———1 Tr rr —r |] "1 oes | | —( rr rT { 1 1} ws [—— 1 rr rr r= rr 1 ( WRSOT [| [rr or 1 1 rr 1 [=] wvswr | | | rr 1 — rr 1 0] wees [© r rr 1 rr 1 1 1 1 wRsw [| 2 rT rr 1 rT 1

Rosy [| 0 | rr 1 rr [1 ]

Rysey 1 rr Tr rr rt rr 1 op sesya | + rr rr tr 1 rr [ csv | rr rr — r+ 1 [~~ vReek [~~ 1 rr rr rr 1 = 1 yess | 2 (rr ~~ rT rr rr 1 rr

Tes ( 0 rr rr rr 1 1 = 1 0] ( resew ( Tr rr rr rr rr pp 0 0] wesw | I 1 rr rT {rr 1 —[ yesw [| rr 7 rT = {1 17 [ veswv [|r 1 1 11 1 0 ] yes [rr rr rr rr 1 1 [1] (wesov [| rr - © 1° [= 1 [ vwsyve | | [| 1 1 [TT 1] *Only 264 out of the 2303 combinations are displayed. None of them were identified in the positive clones.

Table VI: I-Crel variants capable of cleaving the GSCHO1.3 DNA target. pe 33, 38,4044, 68,70,75 and 77 of the I-Crel variants (ex: KRSRES/TYSNI stands for

K28, R30, 832, R33, E38, 540/ T44,

Y68, S70, N75 and 177

Example 3: Making of meganucleases cleaving GSCHO1.4

This example shows that I-Crel variants can cleave the GSCHO1.4

DNA target sequence derived from the right part of the GSCHOI.2 target in a palindromic form (Figure 4). All target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first IT . nucleotides, followed by the suffix P (for example, GSCHOl.4 will be called tggactticgt P).

GSCHOI 4 is similar to STTC_P at positions £1, £2, £3, #4, £5 and +8 and to 10GGA_P at positions 1, +2, £3, +4, £8, £9 and £10. It was hypothesized that positions £6, £7 and +11 would have little effect on the binding and cleavage activity. Variants able to cleave STTC_P were obtained by mutagenesis of I-Crel N75 at positions 44, 68, 70, 75 and 77, as described previously (Arnould ef al., J. Mol.

Biol, 2006, 355, 443-458; Smith er al Nucleic Acids Res., 2006, 34, el49;

International PCT Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO 2007/049156). Variants able to cleave the 10GGA_P target were obtained by mutagenesis of [-Crel N75 or D75, at positions 28, 30, 32, 33, 38, 40 and 70, as described previously in Smith er al. Nucleic Acids Res., 2006, 34, e149;

International PCT Applications WO 2007/060495 and WO 2007/049156.

Both sets of proteins are mutated at position 70. However, the existence of two separable functional subdomains was hypothesized. This implies that this position has little impact on the specificity at bases 10 to 8 of the target.

Therefore, to check whether combined variants could cleave the

GSCHOI 4 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5TTC P were combined with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10GGA_P.

A) Material and Methods a) Construction of target vector :

The experimental procedure is as described in example 2, with the exception that an oligonucleotide corresponding to the GSCHO1.4 target sequence was used: 5° tggcatacaagittitggactitcgtacgaaagtccaacaategtetgtca 3’ (SEQ ID NO: . 185). b) Construction of combinatorial variants

I-Crel variants cleaving 10GGA P or 5TTC _P were previously identified, as described in Smith et al Nucleic Acids Res., 2006, 34, e149;

International PCT Applications WO 2007/060495 and WO 2007/049156, and Arnould et al., J. Mol. Biol, 2006, 355, 443-458; International PCT Applications WO 2006/097784 and WO 2006/097853, respectively for the 10GGA_P and 5TTC_P targets. In order to generate I-Crel derived coding sequences containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5’ end (aa positions 1-43) or the 3’ end (positions 39-167) of the I-Crel coding sequence. For both the 5° and 3° end, PCR amplification is carried out using primers (GallOF 5’-gcaactttagtgctgacacatacagg-3° (SEQ ID NO: 186) or GallOR 5. acaaccttgattggagacttgace-3’ (SEQ 1D NO: 187)) specific to the vector (pCLS1107,

Figure 8) and primers (assF 5’-ctannnttgaccttt-3° (SEQ ID NO: 188) or assR 5’- aaaggtcaannntag-3’(SEQ ID NO: 189)), where nnn codes for residue 40, specific to the I-Crel coding sequence for amino acids 39-43. The PCR fragments resulting from the amplification reaction realized with the same primers and with the same coding sequence for residue 40 were pooled. Then, each pool of PCR fragments resulting from the reaction with primers GallOF and assR or assF and GallOR was mixed in an equimolar ratio. Finally, approximately 25 ng of each final pool of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS1107, Figure 8) linearized by digestion with Dralll and NgoMIV were used to transform the yeast

Saccharomyces cerevisiae strain FYC2-6A (MATa, trpl A63, leu2Al, his3 A200) using a high efficiency LiAc transformation protocol (Gietz and Woods, Methods

Enzymol. 2002, 350, 87-96). An intact coding sequence containing both groups of mutations is generated by in vivo homologous recombination in yeast.

c) Mating of meganuclease expressing clones and screening in yeast .

Screening was performed as described previously (Arnould ef al., J.

Mol. Biol, 2006, 355, 443-458). Mating was performed using a colony gridder (QpixIl, GENETIX). Variants were gridded on nylon filters covering YPD plates, using a low gridding density (4-6 spots/cm®). A second gridding process was performed on the same filters to spot a second layer consisting of the reporter- harboring yeast strain. Membranes were placed on solid agar YPD rich medium, and incubated at 30 °C for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking tryptophan, adding G418, with galactose (2 %) as a carbon source, and incubated for five days at 37 °C, to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02 % X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1 % SDS, 6 % dimethyl formamide (DMF), 7 mM B-mercaptoethanol, 1% agarose, and incubated at 37°C, to monitor B-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software. Positives resulting clones were verified by sequencing (MILLEGEN) as described in example 2.

B) Results

I-Crel combinatorial variants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving STTC_P with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10GGA_P on the I-Crel scaffold, resulting in a library of complexity 1600. Examples of combinatorial variants are displayed in Table VII. This library was transformed into yeast and 3456 clones (2.2 times the diversity) were screened for cleavage against the GSCHO1.4 DNA target (tggactttcgt_P). A total of 250 positive clones were found to cleave GSCHO1.4.

Sequencing and validation by secondary screening of 91 of the best I-Crel variants resulted in the identification of 57 different novel endonucleases. Examples of positives are shown in Figure 9. The sequence of several of the variants identified display non parental combinations at positions 28, 30, 32, 33, 38, 40 or 44, 68, 70, 75, 77 as well as additional mutations (see examples Table VIII). Such variants likely result from PCR artifacts during the combinatorial process. Alternatively, the variants may be I-Crel combined variants resulting from micro-recombination between two original variants during in vivo homologous recombination in yeast.

Table VII: Panel of variants* theoretically present in the combinatorial library

B= | ee positions 44, 68, 70, Amine acids at positions 28, 30,32, 33, 38 and 40

TSand I7 {ex: KRGYQS stands for K28, R30, G32, Y33, 035 and 540) {ex : HNRO! stands for

H44, N68, RTO,

O75and IT? keovas | kesaas | kssas | kestas | kas | KueRas | kaoras | keovas | muas | rss [ wweor — | — J —— 71 — 1 1 1 + TT [— 1 "1 eam [I —71 1 rr Tr 1 — 1 [1 "1 [ gaRDL | + | 1 qe 1 ——1 1 1 : wast [ [1 1 — —71 1 1 1 1 ( ®sw | + "[ —71 — 1 —- 1 © 1 1 wow [~~ 71 [1 1" r wwe | [| 1 7 7 rr 1 1 1 wesw [ 1 Tr 1 rr 1 rr 1 www rr rr—— 1 1 rr rf (esp | + [1 + —7 —71 1 T ]

Wes | + | + J + | + 1 [TT Oe

I oaswe | — [ — [| — 1 +r 71 1 [7 [eww [ —1 —| — 1 71 —1 1 1 T° __ oRewr [1 —T 1 1 = 1 or oem | Oy 1 1 + vr rr T=] ( _opewt | — 1 [1 1 1 71 1 — 1 1] : —aeeew__ | «1 71 rr —71 1 1 oe | —1 — 71 —7T1 —1 1 or =F] — ewrot [+ [+ | TT — 1 1 + | + [0 « (awn | 71 «+ fT" { + __ ®wOl | + [1 —7T —1 1 1 1 =] [ weev [+ [ + [ T1 + 1 [1 {1 1 + 1 * Only 220 out of the 1600 combinations are displayed. + indicates that a functional combinatorial variant cleaving the GSCHO1.4 target was found among the identified positives. 5 .

Table VIII: I-Crel variants with additional mutations capable of cleaving the . GSCHOI1.4 DNA target.

Amino acids at positions 28, 30, 32, SEQ 33, 38, 40 / 44, 68, 70, 75 and 77 ID of the I-Crel variants NO: (ex: KRGYQS/KYSNI stands for

K28, R30, G32, Y33, Q38, S40/

K44, Y68, S70, N75 and I7

KRGYQS/KYSNI

KNSHNS/KNSNI +47K 191

KRGYQS/KNANI +59A : KRSTRS/KNSNI

KRGYQS/KYSNV + 45M

KRGYQS/RYSNI

KNAHQS/KPSNI [196

KRGYQS/KHRDI

KRGYQS/KNRDI

KHRHQS/NYSRY

KRDYQS/QRSRT +80K | 198

KRDYQS/TRSRI +80K 199]

KRGYQS/QYSRY

Example 4: Making of meganucleases cleaving GSCHO1.2 and GSCHO1

I-Crel variants able to cleave each of the palindromic GSCHO1.2 derived targets (GSCHOL.3 and GSCHO1.4) were identified in example 2 and example 3. Pairs of such variants (one cutting GSCHOIL.3 and one cutting

GSCHOI.4) were co-expressed in yeast. Upon co-expression, there should be three active molecular species, two homodimers, and one heterodimer. It was assayed whether the heterodimers that should be formed, cut the GSCHOIL.2 and the non palindromic GSCHOI1 targets.

A) Materials and Methods a) Construction of target vector

The experimental procedure is as described in example 2, with the exception that an oligonucleotide corresponding to the GSCHO1.2 target sequence: 5° tggcatacaagttictgececcagggtacgaaagtccaacaategictgtca 3°(SEQ ID NO: 201) or the

GSCHOI target sequence: 5’ tggcatacaagtttctgccccagggtgagaaagtccaacaategtetgtca 3° (SEQ ID NO: 202) was used. b) Co-expression of variants

Yeast DNA was extracted from variants cleaving the GSCHO1.4 target in the pCLS1107 expression vector using standard protocols and was used to transform E. coli. The resulting plasmid DNA was then used to transform yeast strains expressing a variant cutting the GSCHOL1.3 target in the pCLS0542 expression vector.

Transformants were selected on synthetic medium lacking leucine and containing

G418. c) Mating of meganucleases coexpressing clones and screening in yeast

Mating was performed using a colony gridder (QpixII, Genetix).

Variants were gridded on nylon filters covering YPD plates, using a low gridding density (4-6 spots/cm?). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harboring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated at 30°C for one night, to allow mating, Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, adding G418, with galactose (2 %) as a carbon source, and incubated for five days at 37 °C, to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02 % X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1 % SDS, 6% dimethyl formamide (DMF), 7mM B-mercaptoethanol, 1% agarose, and incubated at 37°C, to monitor [B-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software. :

Bj) Results

Co-expression of variants cleaving the GSCHOIl.4 target (14 variants chosen among those described tn Table VII and Table VIII) and the two variants cleaving the GSCHOL.3 target (described in Table VI) resulted in efficient cleavage of the GSCHO1.2 target in all cases (Figure 10A). In addition, some of these combinations were able to cut the GSCHO! natural target that differs from the

GSCHO1.2 sequence by 2 bp at positions 1 and 2 (Figure 10B). Functional combinations are summarized in Table IX and Table X.

Table IX: Cleavage of the GSCHOQI1.2 target by the heterodimeric variants

Amino acids at positions 28, 30, 32, 33, 38, 40 / 44, 68, 70,75 and 77 : of the I-Crel variants cleaving the GSCHO1.3 target (ex: KRSRES/TYSNI stands for K28, R30, $32,

R33, E38, S40/ T44, Y68, ST), N75 and 177 of CC] KRSRES/TYSNI KRSRES/DYSYQ a =

Cole meee | co

T =

PL oom =n fb; [ooo og =O 13 [moo - on

TE»

FOO E

EE

122] acai

S887

SEG

£2 EE | KRGYQS/IQYSRY + + 28 w 4 55 men

Q& - ar— 0 .

E KRDYQS/QRSRT 3 | KRDYQS/TRSRI + + + 80K + indicates a functional combination

Table X: Cleavage of the GSCHO1 target by the heterodimeric variants

Amino acids at positions 28, 30, 32, 33, 38, 40/ 44, 68, 70,75 and 77 of the I-Crel variants cleaving the GSCHO1.3 target (ex: KRSRES/TYSNI stands for K28, R30, 532,

R33, E38, S40/ T44, Y68, S70, N75 and 177 - KRSRES/TYSNI KRSRES/DYSYQ £ (SEQ ID NO: 184) (SEQ ID NO: 110) ” KRGYQS/RHRDI + ro2 (SEQ ID NO: 130)

EE KRGYQS/KHRDI + = = {SEQ ID NO: 131)

LS KRGYQS/KNRDI +

S$ i 3 {SEQ ID NO: 132)

Ee KRGYQS/NYSRY + v3 & (SEQ [D NO: 203) 30.0 KRGYQS/RTRDI +

SE (SEQ ID NO: 204) a KRGYQS/TYSRV 4+ mee |(SEQIDNO:205)

SS Z5 | KRCYQS/RHRDI +

SESE [GEQIDNO 133) © 8 gw | KRGYQS/KARDI +*

ASHER | (SEQIDNO: 206) 8% KRGYQS/QYSRY in 2.8 < (SEQ ID NO: 200) 2g KRGYQS/NYSRI 2g (SEQ ID NO: 207)

S55 KHRHQS/NYSRY . = 2 (SEQ ID NO: 197) 20% KKSAQS/NYSRY I”

Ss & (SEQ ID NO: 208) £9 KRDYQS/QRSRT

ED + 80K +* & (SEQ ID NO: 198 or KRDYQS/TRSRI + 80K +

SEQ ID NO: 199, + indicates a functional combination *indicates that the combination weakly cuts the GSCHO! targd. 5 Example 5: Improvement of meganucleases cleaving GSCHO1 by random

Lxample >. p g g y mutagenesis of proteins cleaving GSCHO1.3 and assembly with proteins cleaving

GSCHO1.4

I-Crel variants able to cleave the GSCHO1.2 and GSCHOI target by assembly of variants cleaving the palindromic GSCHO!.3 and GSCHO1 .4 target have been previously identified in example 4. However, these variants display stronger activity with the GSCHO1.2 target compared to the GSCHO!1 target:

Therefore the two combinatorial variants cleaving GSCHOL.3 were mutagenized, and variants were screened for cleavage activity of GSCHO1 when co-

expressed with a variant cleaving GSCHO1.4. According to the structure of the [-Crel protein bound to its target, there is no contact between the 4 central base pairs (positions -2 to 2) and the [-Crel protein (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774;

Chevalier ef al., J. Mol. Biol, 2003, 329, 253-269). Thus, it is difficult to rationally choose a set of positions to mutagenize, and mutagenesis was performed on the whole protein. Random mutagenesis results in high complexity libraries. Therefore, to limit the complexity of the variant libraries to be tested, only one of the two components of the heterodimers cleaving GSCHO1 was mutagenized.

Thus, in a first step, proteins cleaving GSCHOL3 were mutagenized, and in a second step, it was assessed whether they could cleave

GSCHOI when co-expressed with a protein cleaving GSCHOI1 .4.

A) Material and Methods a) Construction of libraries by random mutagenesis

Random mutagenesis was performed on a pool of chosen variants, by PCR using Mn**. PCR reactions were carried out that amplify the I-Crel coding sequerice using | the primers preATGCreFor (5- gcataaattactatacttctatagacacgcaaacacaaatacacageggcecttgecace-3’; SEQ ID NO: 209) and ICrelpostRev (5’-ggctcgaggagetegtctagaggatcgetegagitatcagtcggeege-3°; SEQ ID

NO: 210), which are common to the pCLS0542 (Figure 6) and pCLS1107 (Figure 8) vectors. Approximately 25 ng of the PCR product and 75 ng of vector DNA (pCLS0542) linearized by digestion with Neol and Eagl were used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATa, trpl A63, leu2Al, his3 A200) using a high efficiency LiAc transformation protocol (Gietz and Woods,

Methods Enzymol. 2002, 350, 87-96). Expression plasmids containing an intact coding sequence for the I-Crel variant were generated by in vivo homologous recombination in yeast. b) Variant-target yeast strains, screening and sequencing

The yeast strain FYBL2-7B (MAT a, ura3 A851, trp] A63, leu2 Al, lys24202) containing the GSCHOI target in the yeast reporter vector (pCLS1055, : Figure 5) was transformed with variants, in the kanamycin vector (pCLS1107), cutting the GSCO1.4 target, using a high efficiency LiAc transformation protocol. Variant-

target yeast strains were used as target strains for mating assays as described in example 4. Positives resulting clones were verified by sequencing (MILLEGEN) as described in example 2.

B) Results

The two variants cleaving GSCHOI.3, KRSRES/TYSNI and

KRSRES/DYSYQ (I-Crel 30R,33R,38E,44T,68Y,70S,75N and [-Crel 30R,33R,38E,44D,68Y,708,75Y,77Q, also called KRSRES/TYSNI, and

KRSRES/DYSYQ according to the nomenclature of Table VI), were pooled, randomly mutagenized and transformed into yeast. 2304 transformed clones were then mated with a yeast strain that contains (i) the GSCHOI target in a reporter plasmid (ii) : an expression plasmid containing a variant that cleaves the GSCHO1.4 target (I-Crel 30R,32G,44K,68N or KRGYQS/KNRDI according to the nomenclature of Table

VIII). After mating with this yeast strain, 38 clones were found to cleave the GSCHO1 target more efficiently than the original variant. Thus, 38 positives contained proteins able to form heterodimers with KRGYQS/KNRDI with strong cleavage activity for the GSCHOQI target. An example of positives is shown in Figure 11. Sequencing of these 38 positive clones indicates that 19 distinct variants listed in Table XI were identified.

Table XI: Functional variant combinations : displaying strong cleavage activity for GSCHO1.

BN Optimized* Variants GSCHO1.3

SEQ ID NO: 211 to 229

I-Crel 7E 19A 30R 33R 38E 44D 68Y 708 75Y 77Q 120A [-Crel 30R 33R 38E 44D 66H 68Y 70S 75Y 77Q 132V . I-Crel 30R 33R 38E 44D 68Y 708 75Y 77Q 87L ~ = I-Crel 30R 33R 38E 43L. 44D 68Y 703 75Y 77Q r~ a I-Crel 19S 30R 33R 38E 44D 57E 68Y 70S 75Y 77Q 118T 132V [yd ex I-Crel 24V 30R 33R 38E 44T 68Y 705 75N 77T 80K 107R ~~ - Zz I-Crel 30R 33R 38E 44T 50R 68Y 70S 75N tl 5 x 3 I-Crel 30R 33R 38E 39V 44D 68Y 708 75Y 77Q =| & 5 I-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 96R 129A wy =r . ° g 2 I-Crel 30R 33R 38E 44D 45L 50R 68Y 70S 75Y 77Q . 2)

S g g I-Crel 30R 33R 38E 44D 68Y 703 75Y 77Q 107R 129A & : 2% & | 1-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 92R =~ > & I-Crel 30R 33R 38E 44D 68Y 708 75Y 77Q 161P ’ 2 I-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 120E = I-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 87L 139R ay ¥ I-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 105A

I-Crel 30R 33R 38E 44D 64A 68Y 705 75Y 77Q 871 105A 117V 137N

I-Crel 24V 30R 33R 38E 44D 68Y 70S 75Y 77Q

I-Crel 30R 33R 38E 44T 68Y 70S 75N 132V * Mutations resulting from random mutagenesis are in bold. ) 5 Example 6: Improvement of meganucleases cleaving GSCHOI1 by site-directed mutagenesis of proteins cleaving GSCHOI1.3 and assembly with proteins cleaving

GSCHO1.4

The initial [-Crel variants cleaving GSCHOI.3 described in

Table VI and used for random mutagenesis in example 5 were also mutagenized by introducing selected amino-acid substitutions in the proteins and screening for more efficient variants cleaving GSCHOI in combination with a variant cleaving

GSCHO1 4.

Six amino-acid substitutions have been found in previous studies to enhance the activity of I-Crel derivatives: these mutations correspond to the replacement of Glycine 19 with Serine (G19S), Phenylalanine 54 with Leucine (F54L), Glutamic acid 80 with Lysine (E80K), Phenylalanine 87 with Leucine (F87L),

Valine 105 with Alanine (V105A) and Isoleucine 132 with Valine (I1132V). These mutations were individually introduced into the coding sequence of proteins cleaving

GSCHOL.3, and the resulting proteins were tested for their ability to induce cleavage of the GSCHOI| target, upon co-expression with a variant cleaving GSCHO1.4,

A) Material and Methods a) Site-directed mutagenesis

Site-directed mutagenesis libraries were created by PCR on a pool of chosen variants. For example, to introduce the G19S substitution into the coding sequence of the variants, two separate overlapping PCR reactions were carried out that amplify the 5” end (residues 1-24) or the 3’ end (residues 14-167) of the I-

Crel coding sequence. For both the 5’ and 3° end, PCR amplification is carried out using a primer with homology to the vector (Gall OF 5’-gcaactttagtgctgacacatacagg-3’ (SEQ ID NO: 186) or GallOR 5’-acaaccttgattggagacttgacc-3’(SEQ ID NO: 187)) and a primer specific to the I-Crel coding sequence for amino acids 14-24 that contains the substitution mutation G19S (G19SF 5’-gecggctitgtggactctgacggtageateate-3° (SEQ ID

NO: 230) or GI9SR 5’-gatgatgctaccgtcagagtccacaaagecgge-3° (SEQ ID NO: 231).

The resulting PCR products contain 33bp of homology with each other. The PCR fragments were purified. Approximately 25ng of each of the two overlapping PCR fragments and 75ng of vector DNA (pCLS0542, Figure 6) linearized by digestion with Ncol and Eagl were used to transform the yeast Saccharomyces cerevisiae strain

FYC2-6A (MATa, trpl A663, leu2 Al, his3A200) using a high efficiency LiAc transformation protocol (Gietz and Woods, Methods Enzymol., 2002, 350, 87-96).

Intact coding sequences containing the G19S substitution are generated in vivo homologous recombination in yeast.

The same strategy is used with the following pair of oligonucleotides to create other libraries containing the F54L, E80K, F87L, V105A and 1132V substitutions, respectively: * F54LF: 5’-acccagegeegtiggctgetggacaaactagtg-3’ and F54LR: 5°- cactagtttgtccagcagecaacggegetgggt-3’ (SEQ ID NO: 232 and 233);

E80KEF: 5’-ttaagcaaaatcaagccgetgeacaacttectg-3’ and E80KR: 5- caggaagitgtgcageggcttgattttgettaa3’ SEQ ID NO: 234 and 235);

* FR7LF: 5”-aagcecgetgeacaacctgetgactcaactgeag-3’ and F87LR: 5’- ctgcagttgagtcagcaggttgtgcageggcett3’ SEQ ID NO: 236 and 237); * VI05AF: 5’-asacaggeaaacctggetetgaaaattatcgaa-3° and ~VI0O5AR: 5°- ttegataattttcagagccaggtttgectgttt-3° SEQ ID NO: 238 and 239); * T1132VF: 5-acctggptggateaggtigeagcictgaacgat 3’ and [132VR: 5’- atcgttcagagetgcaacctgatccacccaggt-3° SEQ ID NO: 240 and 241). c)} Mating of meganuclease expressing clones and screening in yeast

The experimental procedure is as described in example 5. d) Sequencing of variants | :

The experimental procedure is as described in example 2.

B) Results

Libraries containing one of six amino-acid substitutions (Glycine 19 with Serine, Phenylalanine 54 with Leucine, Glutamic acid 80 with Lysine,

Phenylalanine 87 with Leucine, Valine 105 with Alanine and Isoleucine 132 with

Valine) were constructed on a pool of two variants cleaving GSCHOL.3

KRSRES/TYSNI and KRSRES/DYSYQ (I-Crel 30R,33R,38E,44T,68Y,70S,75N and I-Crel 30R,33R,38E,44D,68Y,708,75Y,77Q, also called KRSRES/TYSNI, and

KRSRES/DYSYQ, respectively, according to the nomenclature of Table VI}. 192 transformed clones for each library were then mated with a yeast strain that contains (i) the GSCHOI target in a reporter plasmid (ii) an expression plasmid containing a variant that cleaves the GSCHO1.4 target (I-Crel 30R,32G,44K,68N or

KRGYQS/KINRDI) described in example 3.

After mating with this yeast strain, a large number of clones (>20} in each of the libraries, except for the library containing amino-acid substitution

Phenylalanine 54 with Leucine, were found to cleave the GSCHO!1 target more efficiently than the original variants. An example of positives is shown in Figure 12.

The sequence of the five best I-Crel variants cleaving the GSCHO1 target when forming a heterodimer with the KRG YQS/KNRDI variant are listed in Table XII.

Table XII: Functional variant combinations displaying strong cleavage activity for GSCHOI.

CT ee

SEQ ID NO: 242 to 244, 213, 226 = S== 8 g 5 8 I-Crel 19S 30R 33R 38E 44D 68Y 70S 75Y 77Q 2 3 3 & © | 1-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 132V = 2 5 I-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 87L z ® Hh = [-Crel 30R 33R 38E 44D 68Y 70S 75Y 77Q 105A * Mutations resulting from site-directed mutagenesis are in bold.

Example 7: Improvement of meganucleases cleaving GSCHOL by random mutagenesis of proteins cleaving GSCHO1.4 and assembly with protcins cleaving

GSCHOL1.3

As a complement to example 4 we also decided to perform random mutagenesis with variants that cleave GSCHO1.4. The mutagenized proteins cleaving GSCHOI1.4 were then tested to determine if they could efficiently cleave GSCHOI1 when co-expressed with a protein cleaving GSCHO1.3.

A) Material and Methods - a) Construction of libraries by random mutagenesis :

Random mutagenesis was performed on a pool of chosen variants, by PCR using Mn™". PCR reactions were carried out that amplify the I-Crel coding sequence using the primers preATGCreFor (5°- geataaattactatactictatagacacgcaaacacasatacacageggecttgccace-3’; SEQ ID NO: 209) and ICrelpostRev (5-ggctcgaggagetegtetagaggategetegagttatcagteggeege3'; SEQ ID

NO: 210). Approximately 25 ng of the PCR product and 75 ng of vector DNA (pCLS1107, Figure 8) linearized by digestion with Dralll and NgoMIV were used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATa, trpl 263, leu2 Al, his3.A200) using a high efficiency LiAc transformation protocol (Gietz and

Woods, Methods Enzymol., 2002, 350, 87-96). Expression plasmids containing an intact coding sequence for the I-Crel variant were generated by in vive homologous recombination in yeast.

b) Variant-target yeast strains, screening and sequencing

The yeast strain FYBL2-7B (MAT a, ura3 A851, trpl A63, leu? Al, lys2.A202) containing the GSCHOI target in the yeast reporter vector (pCLS1055,

Figure 5) was transformed with variants, in the leucine vector (pCLS0542), cutting the

GSCHOL.3 target, using a high efficiency LiAc transformation protocol. Variant- target yeast strains were used as target strains for mating assays as described in example 4. Positives resulting clones were verified by sequencing (MILLEGEN) as described in example 2.

B) Results

Nine variants cleaving GSCHO1.4 (I-Crel 30R,32G,44K,68Y,708,75N, I-Crel 33H,38N,44K,47K,68N,70S,75N, . [-Crel 30K,33A,75N, I-Crel 30R,32G,44K,59A,68N,70A,75N, I-Crel 30R,33T,38R,44K,68N,70S,75N, I-Crel 30R,32G,44K,45M,68Y,70S,75N,77V, I-

Crel 30R,32G,44K,68N,70S,75N, I-Crel 30R,32G,44R,68Y,70S,75N and I-Crel 32A,33H,44K,68P,708,75N also called KRGYQS/KYSNI, KNSHNS/KNSNI +47K,

KKSAQS/QRRNI, KRGYQS/KNANI + 59A, KRSTRS/KNSNI, KRGYQS/KYSNV + 45M, KRGYQS/KNSNI, KRGYQS/RYSNI and KNAHQS/KPSNI, respectively, according to the nomenclature of Table VII and Table VIII) were pooled, randomly mutagenized and transformed into yeast. 4608 transformed clones were then mated with a yeast strain that contains (i) the GSCHOI target in a reporter plasmid (ii) an expression plasmid containing a variant that cleaves the GSCHO1.3 target (I-Crel 30R,33R,38E,44D,68Y,708,75Y,77Q or KRSRES/DYSYQ according to the nomenclature of table VI). After mating with this yeast strain, 254 clones were found to cleave the GSCHOI target more efficiently than the original vanants. Thus, 254 positives contained proteins able to form heterodimers with KRSRES/DYSYQ with strong cleavage activity for the GSCHOI1 target. An example of positives is shown in

Figure 13. Sequencing 32 of the strongest positive clones indicates that 18 distinct variants listed in Table XIII were identified.

Table XJII: Functional variant combinations displaying strong cleavage activity for GSCHO1.

Optimized Variants GSCHO1.4

SEQ ID NO: 245 to 262)

I-Crel 3A 30R 33R 68A 75D 77R

I-Crel 30R 32G 68A 75D 77R 119L

I-Crel 19S 30R 32D 44R 68H 75D 161T g I-Crel 30R 32G 44R 68H 75D 132V 154G @ | 1-Crel 28 30R 33H 68A 75D TTR € | 1-Crel 30R 33R 68A 75D 77R “© 5 _. | I-Crel 30R 33H 68A 75D 77R e 3 = I-Crel 30R 32G 44R 68H 75D 1251 132V 160R } 2 SB | -Crel 30R 33H 68A 75D 77R 114F

E 2 a I-Crel 12H 30R 32A 33H 45M 68S 75D 77R = 2 g I-Crel 30R 33H 60Y 68A 75D 77R $ | = | 1-Crel 30R 33H 50R 68A 75D 77R ’ 3 I-Crel 30R 33H 68A75D 77R 110V 153N & I-Crel 6K 30R 33H 68A 75D 77R 114P — & | I-Crel 30R 33H 35L 68A 75D 77R = I-Crel 30R 32G 33H 68S 75D 77R 137Y :

I-Crel 30R 33H 38H 68A 75D 77R ‘

I-Crel 30R 33H 68T 75D 77R * Mutations resulting from random mutagenesis are in bold. ** Variants are derived from the FCref N75 scaffold and position 75 was mutated to aspartic acid (D) during cycle of random mutagenesis.

Example 8: Improvement of meganucleases cleaving GSCHO1 by site-directed mutagenesis of proteins cleaving GSCHOL1.4 and assembly with proteins cleaving ‘GSCHOL.3

The initial I-Crel variants cleaving GSCHO1.4 described in Tables 3 and 4 and used for random mutagenesis in example 7 were also mutagenized by introducing selected amino-acid substitutions in the proteins and screening for more efficient variants cleaving GSCHO! in combination with a variant cleaving

GSCHOI1.3.

Six amino-acid substitutions have been found in previous studies to enhance the activity of I-Crel derivatives: these mutations correspond to the replacement of Glycine 19 with Serine (G19S), Phenylalanine 54 with Leucine (F54L), Glutamic acid 80 with Lysine (E80K), Phenylalanine 87 with Leucine (F87L),

Valine 105 with Alanine (V105A) and Isoleucine 132 with Valine (I1132V). These mutations were individually introduced into the coding sequence of proteins cleaving

GSCHOI.3, and the resulting proteins were tested for their ability to induce cleavage of the GSCHOI target, upon co-expression with a variant cleaving GSCHOI 4.

A) Material and Methods a) Site-directed mutagenesis

Site-directed mutagenesis libraries were created by PCR on a pool of chosen variants. For example, to introduce the G19S substitution into the coding sequence of the variants, two separate overlapping PCR reactions were carried out that amplify the 5” end (residues 1-24) or the 3” end (residues 14-167) of the I-

Crel coding sequence. For both the 5” and 3° end, PCR amplification is carried out using a primer with homology to the vector (GallOF 5’-gcaactttagtgctgacacatacagg-3’ or GallOR 5’-acaaccttgattggagacttgace-3’) and a primer specific to the -Crel coding sequence for amino acids 14-24 that contains the substitution mutation G19S (G19SF 5’-gccggctttgtggactctgacggtageateate-3° (SEQ ID NO: 230) or GI9SR 5’- gatgatgctaccgtcagagtccacaaageegge-3’ (SEQ ID NO: 231)). The resulting PCR > products contain 33bp of homology with each other. The PCR fragments were purified. Approximately 25ng of each of the two overlapping PCR fragments and 75ng of vector DNA (pCLS1107, Figure 8) linearized by digestion with Dralll and

NgoMIV were used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MAT, trpl A63, leu2 Al, his3.A200) using a high efficiency LiAc transformation protocol (Gietz and Woods, Methods Enzymol., 2002, 350, 87-96). Intact coding sequences containing the G19S substitution are generated by in vivo homologous recombination in yeast.

The same strategy is used with the following pair of oligonucleotides to create other libraries containing the F54L, E80K, F87L, V105A and [132V substitutions, respectively: * F54LF: 5’-acccagegecgtiggetgetggacaaactagtg-3° and FS54LR: 5 cactagtttgtccagcagecaacggegetgggt-3’ (SEQ ID NO: 232 and 233); * E80KF: 5’-ttaagcaaaatcaagccgctgeacaactteetg-3’ and EBOKR: 5’- caggaagttgtocageggcttgattttgettaa-3’ SEQ ID NO: 234 and 235); ’

* F87LF: 5’-aagccgetgeacaacctgetgactcaactgeap-3’ and F87LR: 5’- ctgeagttgagtcageaggttgtgeageggett-3’ SEQ ID NO: 236 and 237); * VIO5AF: 5’-aaacaggcaaacctggctctgaaaattatcgaa-3’ and VI105AR: 5'- ttcgataattttcagagccaggtttgcctgttt-3° SEQ ID NO: 238 and 239); *¥ [I132VF: 5’-acctgggtggatcaggttgcagetctgascgat3™ and [132VR: 5°- atcgttcagagctgeaacctgatccacccaggt-3’ SEQ ID NO: 240 and 241). c) Mating of meganuclease expressing clones and screening in yeast

The experimental procedure is as described in example 7. d) Sequencing of variants

The experimental procedure is as described in example 2.

B) Results

Libraries containing one of six amino-acid substitutions (Glycine 19 with Serine, Phenylalanine 54 with Leucine, Glutamic acid 80 with Lysine,

Phenylalanine 87 with Leucine, Valine 105 with Alanine and Isoleucine 132 with

Valine) were constructed on a pool of nine variants cleaving GSCHOI1.4 (I-Crel 30R,32G,44K,68Y,708,75N, [-Crel 33H,38N,44K,47K,68N,70S,75N, I-Crel 30K,33A,75N, I-Crel 30R,32G,44K,59A,68N,70A,75N, I-Crel 30R,33T,38R,44K,68N,708,75N, I-Crel 30R,32G,44K,45M,68Y,708,75N,77V, I-

Crel 30R,32G,44K,68N,708S,75N, [-Crel 30R,32G,44R,68Y,70S,75N and I-Crel 32A,33H,44K,68P,70S,75N also called KRGYQS/KYSNI, KNSHNS/KNSNI +47K,

KKSAQS/QRRNI, KRGYQS/KNANI + 59A, KRSTRS/KNSNI, KRGYQS/KYSNV + 45M, KRGYQS/KNSNI, KRGYQS/RYSNI and KNAHQS/KPSNI, respectively, according to the nomenclature of Table VII and Table VIII). 192 transformed clones for each library were then mated with a yeast strain that contains (i) the GSCHOI target in a reporter plasmid (ii) an expression plasmid containing a variant that cleaves the GSCHOI1.3 target (I-Crel 30R,33R,38E,44D,68Y,708,75Y,77Q or

KRSRES/DYSYQ) described in example 2.

After mating with this yeast strain, a large number of clones (>20) were found to cleave the GSCHO1 target more efficiently than the original variants for the libraries containing amino-acid substitution Glycine 19 with Serine,

Phenylalanine 54 with Leucine and Isoleucine 132 with Valine. An example of positives is shown in Figure 14. The sequence of the two best [-Crel variants from each library cleaving the GSCHOI target when forming a heterodimer with the

KRSRES/DYSYQ variant are listed in Table XIV. These variants display non parental combinations at positions 28, 30, 32, 33, 38, 40 or 44, 68, 70, 75, 77. Such combinations likely result from PCR induced mutations during the combinatorial process.

Table XIV: Functional variant combinations displaying strong cleavage activity for GSCHOI.

I EC: Cl

SEQ ID NO: 263 to 268 “ & 2 : 3 4 o I-Crel 19S 30R 33H 638A 75D TTR

O _ 3 > s I-Crel 19S 30R 32G 44K 45M 68H 75D

Z = 2 > & | I-Crel 30R 32G 44R 68H 75D 132V 2 2 8 g 1-Crel 30R 32G 44R 54L 68H 75D > & I-Crel 30R 33T 38R 44K S4L 68H 75D * Mutations resulting from site-directed mutagenesis are in bold. ** Variants are derived from the FCref N75 scaffold and position 75 was mutated to aspartic acid (D) during improvement. }

Example 9: Validation of GSCHO1 target cleavage in an extrachromosomal model in CHO cells

I-Crel variants able to efficiently cleave the GSCHOLI target in yeast when forming heterodimers were described in examples 4, 5, 6, 7 and 8. In order to i identify heterodimers displaying maximal cleavage activity for the GSCHOI target in

CHO cells, the efficiency of chosen combinations of variants to cut the GSCHOI target was compared, using an extrachromosomal assay in CHO cells. The screen in

CHO cells is a single-strand annealing (SSA) based assay where cleavage of the target by the meganucleases induces homologous recombination and expression of a LagoZ reporter gene (a derivative of the bacterial lacZ gene). 1) Materials and methods ‘ a) Cloning of GSCHOI target in a vector for CHO screen

The target was cloned as follows: oligonucleotide corresponding to the GSCHOI target sequence flanked by gateway cloning sequence was ordered from

PROLIGO: 5° tggcatacaagtttctgccccagggtgagaaagtccaacaategtetgtea 3° (SEQ ID NO: 202). Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into CHO reporter vector (pCLS1058, Figure 15). Cloned target was verified by sequencing (MILLEGEN). b) Re-cloning of meganucleases

The ORF of I-Crel variants cleaving the GSCHO1.3 and GSCHO! .4 "targets identified in examples 3, 5, 6, 7 and 8 were re-cloned in pCLS1768 (Figure 16). ORFs were amplified by PCR on yeast DNA using the attBl-ICrelFor (5°- ggppacaapttiptacaaaaaapcaggcttcgaaggagatagaaccatggecaataccaaatataacaaagagtics 37;

SEQ 1D NO: 269) and attB2-ICrelRev (5°- ggggaccactitgtacaagaaagcetggptitagtcggcegeegggpaggatitetictictege3’; SEQ ID NO: 270) primers. PCR products were cloned in the CHO expression vector pCLSI768 (Figure 16) using the Gateway protocol (INVITROGEN). Resulting clones were verified by sequencing (MILLEGEN). c) Extrachromosomal assay in mammalian cells

CHO cells were transfected with Polyfect® transfection reagent according to the supplier’s protocol (QIAGEN). 72 hours after transfection, the level of Beta galactosidase expression for each transfection was quantified using the Beta-

Glo® Assay System (Promega). The Beta-Glo Assay contains a luciferin-galactoside substrate (6-O-B-galactopyranosylluciferin) that can be cleaved by p-galactosidase to form luciferin that is then utilized in a firefly luciferase reaction to generate light. For each transfection, approximately 100,000 cells in 100pl of medium were combined with an equal volume of Beta-Glo lysis/revelation buffer as described by the manufacturer (Promega). After 30 minutes of incubation at room temperature, signal was measured with a luminometer (Perkin Elmer Victor multilabel plate reader).

Per assay, 150 ng of target vector was cotransfected with 25 ng of each one of both variants (25 ng of variant cleaving palindromic GSCHOI1.3 target and 25 ng of variant cleaving palindromic GSCHOI .4 target). 2) Results

Several variants described in examples 3, 5, 6, 7 and 8 were first re- cloned in pCLS1768 (Figure 16). Then, in order to identify the heterodimer displaying the maximal cleavage activity with the GSCHOL1 target in CHO cells, I-Crel variants cleaving the GSCHO1.3 or GSCHO/ .4 targets (described in examples 3, 5, 6, 7 and 8)

were tested together as heterodimers against the GSCHO! target in the CHO extrachromosomal assay.

Figure 17 shows the results obtained for 12 heterodimers tested and the values of the different combinations are compiled in Table XV. Analysis of the efficiencies of cleavage of the GSCHO1 sequence demonstrates that 10 of the 12 combinations of I-Crel variants are able to efficiently cut the GSCHOI target in CHO + cells. ‘

Table XV: Functional heterodimeric combinations cutting the GSCHOI target in CHO cells. 10 .

Optimized variants cleaving GSCHO1.3 . Mt 3C

Mt 3A Mt 3B19A 30R 33R 38E 19S 30R 33R 38E 44D 57E 30R 33R 38E 44D 66H 44D 68Y 70S 75Y 77Q 68Y 70S 75Y 77Q 118T 68Y 70S 75Y 77Q 132V 120A 132v {SEQ ID NO: 212) (SEQ ID NO: 271} {SEQID NO: 215)

Mt 4A : 1895 30R 32G 44K 2.8x 108 0.04 x 108 45M 68H <« | (SEQID o NO: 264 }

S| MB éG | 30Rr32G = 68A TTR 23x 108 3.1 x108 2.8x 106 = 119L 2 | (SEQID a NO: 246 8 [mac :

S | 30R33R ; 7 688A 77R 311x108 2.9x10 ¥ (SEQ = | NO: 250 o Mt 4D i 30R 326 . ; 44R 68H 25x10 31x10 (SEQ ID

NO: 130 i s1546PCT25.5T25. txt

SEQUENCE LISTING

<110> CELLECTIS

SMITH, Julianne <120> Meganuclease variants cleaving a DNA target from a glutamine synthetase gene and uses thereof <130> 1546PCT25 i <160> 272 <170> PatentIn version 3.3 <210> 1 . <211> 163 <212> PRT <213> Cchlamydomonas reinhardtii ’ ‘<400> 1

Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe 1 5 10 15 val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser

Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn val Thr Gln Lys 40 - 45

Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly val 50 55 60

Gly Tyr val Arg Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser Glu 65 70 75 BO

Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu Lys 85 90 95

Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln Leu 100 105 110

Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr Trp 115 120 125 - val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr 130 135 140

Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys Lys 145 150 155 160

Ser Ser Pro <210> 2 <211> 24 <212> DNA . Page 1

S1546PCT25.5T25. txt oo <213> ARTIFICIAL SEQUENCE <220> <223> (C1221 DNA target <400> 2 tcaaaacgtc gtacgacgtt ttga 24 <210> 3 <211> 9770 <212> DNA <213> Mus musculus <400> 3 cagagcggag aatgggagta gagcagagtg tctgaacagc acgctcaccc atctcctctc 60 cgcctegete tcctgacctg ttcacccatc catcatccgg ccggccaccg ctctggtaag 120 cgcacggagg gtccaggaat gtacggccygc ccgggctgtg gggtcgcact ctacttccca 180 gctccagtca gctgctgtac ggagcgggat gcagcgctcg tgectcccgc gtgtttgcag 240 cgtgcggcec gggccggcete tgcttggtgt tcctaggacg cgoctgtgagc caagctccgg 300 gaagggcggg gttgcgggtt gttttgatct gttctatact tgcggccgga ggecgecgecce 360 cgggaggcag gcgccgttgg ctggygttca cgetgagatg ggggctttcc ggtggtccca 420 gcgggagagg gttcttgect taggtgggcg caggcgcctt gatcctctct tcctggcgge 480 gcatttgggg ggcgtcgtca cgctgtgggt ggtctggttg aggatggtgg tcctaagegt 540 tgatggcacc actccccage tcccaacgcc gtgtcctagg cctttaccat atgaccgaac 600 aatggagagc cggagccccg gagtggecgg cgggctccgc agtggagagg ccgcgccaag 660 cggaggcagc agcgycgege tgtgcctccce geggtegece cactcetegec acccggectce 720 taccctcgec ggggtatggce cccctgggag aggccttgag atctacgcgg gcccgagggt 780 cgcggcaccg actttccgga cattttagtg ggaaggctge tttcaaagtg gattgcccca © 840 actcctccgg gggcgggaag cggggatcct cccccagccg caaatactca aagaaaccaa 900 ccattgaaga cgtagaagat ggagattctc ggtcctcaga gtccccttct aatactttag 960 gcttegttyge ctactctgtg aactccgggg agaagtcgag ggttaagatt aaatcgcacc 1020 cgtcttattc cagcacctcc ccctccgaac ggtetgggtc ccccactcca tcgecctegce 1080

CCaaaaagct ccgttgctta gaccagcgag aaatcgagaa cgaggagagg catgaacact 1140 gctctaaaaa gaggaggtct agagagtaca accccagtgc atttgattct cattggctgg 1200 ggtgagtaaa agtcagggcg aaggaccccg ggtgcatctg gcaacccgca gaaactactc 1260 agaattttaa gaacccattc cactttgcac tacagggaca ccagttggtg ctatctatgt 1320 acactaggct gctggcaccc agctggtctc agggaaccag ggccagagga ggaactcagg 1380 ttcccctaac agttcattaa tgctggatgt gtgtgtgtgg ggggeggyggg gcacggcaga 1440 ggaggaaagc tgatgagtgg tgtaaattga agccatctag aattacaatc cgggcctcta 1500 agtggtgtag gcagaggctt tggttctgca tcggacttga cagcagagge tcatctgttc 1560 cccyggggaa gggtgaggct tttggaggga gaggcagttg tittcacttg ggcaaacatg 1620

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$1546PCT25.5T25. txt gacggttgcc catagaaact ttgccactgt acttcagaaa gttgcccaag tcattggagg 1680 dgaacaatat gttccctctc cagctatccg gggagattag ggagggaggg gggcatttcc 1740 tcttgtgttt tgaggctggt ttittgtagec ctcacattca tcgaagatct tgccccgact 1800 cctgtggctt gtaaacttag aagtgggatt tttctacgta ccagaaaata ttatccggtg 1860 ggatcgtcaa atactgttaa ttttcacatt gaaatctgtc tctggagtaa ggcttttcac 1920 cacagtaatg aagtcagcag ttgaggcctg gtgtggaggg ggcatacctt taatcccagce 1980 tctcaggagg cagagactgg tagatcctct gattttgagg aaagcttggt gtaaataatg 2040 agtaagtaat tactaggaca gagagagggc ttgttgagat ttctctgtct caaaaaaaaa 2100 daaaaaaaacC Caaaaaacca gtcagcagtg tgctccttgg agtatggctt tactttttac 2160 caccttagtg gtggcagata atacaggtcc tttctttctc ccaggaatat gttageccttt 2220 aaagtcttac agctagccct agtatttcat tgtgtaatct aaagatggtg gtggcataca 2280 cCtttaattc tagcgttcag gatgctgaga ggggcagatc taatttgatt ctgaggctag 2340 cctggtcggc ataagttcca gggctccaca atgagacttt gtctcaaata agtagatagt 2400 ctaaatgtag gtatgtaggg attacaatgg gttytgtgtc ccttaaggtt gttccaagag 2460 ctgagcggtg gtggcacatg cctttattta atcccagcac ttgggaggca gaggcaggtg 2520 gatttctgag ttcaaggcca gcctggtcta caaagtgagc tccaggacaa ccagggccat 2580 tcagagaaac cctgtctcca aaaaaaaaaa aaaaggctgt tccaaagagt ggaagacaaa 2640 gcaagactca acagtcaatt agtcaagtict ttctgtggca ataaggtagt tctgttggta 2700 gaataagata ttctgttaat gaacgtcttg atatttgttc tctcctgcta acatttctca 2760 agtttaacgg ttattaaaat cccttaacta gtccccctga gggggcatgg tccttgtcta 2820 atataaaact ttaaacccct tgaaagcaga gtgaataatg cacctttgtg tgtgtccagt 2880 cccagaggag cgagatcacc acgcacgcca gcctgattce cttgecgtec cctacagtgg 2940 atccctttga attcgatatt aataaaatgc gatttctgtc tctctccaga acaccttcca 3000 ccatggccac ctcagcaagt tcccacttga acaaaggcat caagcaaatg tacatgtcce 3060 tgccccaggg tgagaaagtc caagccatgt atatctgggt tgatggtacc ggagaaggac 3120 tgcgctgcaa gacccgtace ctggactgtg agcccaagtg tgtggaaggt gagtgccggg 3180 gcggagtgty cgcacgcctg ggagtgtacg cacagcctcg gatccacctt ccttctgttt 3240 ggtttgcaag gcttticaga ccttagtcag tcacccgtaa gtaagctgct gcatagtctg 3300 gaggcgcagc aacaatggaa gocctttcttt agatggactc tggcgtgtgc tggtacattg 3360 aagaaaaata ctgggtcacg tttgtggggg atgggaggct gctgtgtgct aacctggcca 3420 accccaggaa cctagtttga gaggactggt gtaactggaa tatgctatct agtttataga 3480 acagtcggtc tcaacccttc ctaatgcttc caccatttaa taaggctagt gttgtggcaa 3540 ccaccaacca taaaatggtt tttgccgcta ctttataatt gtatatactt ctaagtttat 3600 tttagttgta gcctgtgttt ctcagcatag accaagtaag ccatagctgc tcaggagagg 3660

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51546PCT25.5T25. txt ggtggcccce tcaccatcta cctggcttag gatgggttac tcttccaagg atgtttctgt 3720 ttgagtgaac gagtgaccag ataacagagc atggattgta tacttggtac ttggcagagt 3780 gtgggtaggt tcttcagtct ctgctttctg agaactcaga ggtaactgga gagtcaaacc 3840 cgaccactaa gacagtaggg aaaagaccaa gcaagcggtg gggaagcaac tgtttatata 3900 caagcataac ttgaagtaca acagttggac ctgtggggaa taggagaagg gagatgatga 3960 tggcctagag gaggaggtgg ggtttttttg tttgttttgg gttttgtcct gtgtgtgtct 4020 gacacactgg aagtgatacc agagtacaca ggagacttgc acagagagga ctggttttct 4080 gctcaggtgg ctcttgggat gcagtgctct ggggactctc aggtcaggag aaactgggat 4140 aggtgacagc gataggtgac gtcaggttat ccctgatgta gatgcagtca catgcacctc 4200 actcctcata aagacaaagt ggtggtgagc aacgaaagga tgagtaagag gctgaccgtc 4260 ttcttagcgt gtgcactaaa aacttttaaa actgtatgta tatgggtgtg tatgtgcgcg 4320 cgcgcacgcg cgctccatgt gctcagtgcc agcagaaatc aaagagtgag gaccgcagga 4380 actgaagtta cagacagttg tgggctttca tgtgattgag agctcttgtt gctctcaccg 4440 atctgggttc agttaacaac acccacatag tggctcacaa ccatctgtaa ctctcaattc 4500 cgggagatcc aaccctcctc gtctggcttc ttcctgcagg ctcaaataac acgtttaaag 4560 ttaatttaat tttctatctt tgtcttgccc agagttacct gagtggaact ttgatggctc 4620 tagtaccttt cagtctgaag gctccaacag cgacatgtac ctccatcctg ttgccatgtt 4680 tcgagacccc ttccgcaaag accccaacaa gcetggtgcta tgtgaagttt tcaagtataa 4740 ccggaaacct gcaggcaagt atgggatggg tgtggctggce tgtaaatcct gaaactctag 4800 gygaggtgaca ttctcaatga attgagaagc cgctctctaa gaacgtaggg atggcagggt 4860 ggctattcta ccctgaactt tcctgttggg aacagtgtgc tcaatccttt cttccatggt 4920 tccttttatt gtttgtatag tgttggctct tctgtctygtt tgctgacggg caacctctac 4980 ctgctgatct aaaagcctgt gtttaaaagt tctgtagttt ttgaatttaa atactagatc 5040 taccactgtt ctacctgctt ttttttcttc tgaattgtgt gtgctgtatg tggagagcat 5100 gcgtcggaga gcatgccttg ctgggtgcgt ggacgtcaac gggcagcgtt ggagttggtt 5160 gtctcctgee tgtttatgtg agctctggag gtggaactca ggttgccagg ctcgagtggc 5220 aacaccttta cccactgagc cgtctcagta gtcttctctg ataagagccc atcccgaagt 5280 cattggaaga tcacatgaat gaccgtgtgc cacaatcact tgggagtact gcacgttaac 5340 tatcggttac tattttatgc cagacagctt gttgagtctg aatactcaca agtatgagca 5400 tcgagacttc gggatgtgca tttaccctgc ctgcacatgc gtggagttta gtttgcactg 5460 tgctatccat ctcecgttttc agatagcttt gaacctgggg aagtctcact tctgtgaaat 5520 atcttctagc aatgccagta aggcttggtg gccctgggcc ttcagtgctt ctgtttcaaa 5580 aggcagtagc attattggta aaaggtgtgc tctgtgcctg tcctgtcacc tgggtgtgcc 5640 acctaacacc ctctcttggg gtttcatttt cttatttgtg aaaatgaagg tttttttggt 5700

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— Lo s1546PCT25.5T25. txt ttgttttttt ttgttttttt ttttccaaga cagagtttct ctgtgtagcc ctggctgtcc 5760 tggaactcac tctgtagacc aggctggcct tgaactcaga aatctgcctg cctctgectc 5820 ccaagtgctg ggatcaaagg tgtgcgccac catgccctgce caaaatgaag gttgttaata 5880 ccttagactc agatggttat tttttttcta gcttgggaat tgttttcagc tatactcata 5940 tataattata tgtctgtatg tctgtctacc tatctacttt tgtaccagtc ataactgtaa 6000 aacttagcac ttaaactcac ctggcatgtg aacctaatgg acaaattatt ctcaataaca 6060 gatctggecct tcagtgtctg agacactagg gaatacatct gacaactaga agcagttgtc 6120 ctgtgaatct gagaatgagt gcctggcgtg tggtgttggg atggtggcca gcatgcagat 6180 agggtgacca cttgctcgga ttccattccc atgatgctgc gtggctgact ttaattataa 6240 aaaagtctat tagccatttc ctgcaaatgg acaattatca cttcttccat tttccttggt 6300 gataaaacat ggtggttagg cctgggtcag tctctcctgt gaccagcact ggagctgtgt 6360 gagagggctc tagtcctgat ggcatgttga ttctttattt ctagagacca acttgaggca 6420 catctgtaaa cggataatgg acatggtgag caaccagcac ccctggtttg gaatggagca 6480 ggaatatact cttatgggaa cagacggcca cccatttggt tggccttcca atggcttccc 6540 tggaccccaa ggtacgtccc actgggtaaa gggtgaaact tcctccccta agttgttact 6600 gtccaggaaa tccccttccc cagagatagg tgcaatcctg aaatgagaaa atggagacca 6660 gcagcagaat cttaacagta gaccgacctt gcatccctca catccagagg tggttagaat 6720 ttaaagtgac agagagtggt gagatggctc agtggttacc caccaggcct gacaacctga 6780 atctcctcct gggacccatt tggtagaagg caagaaatga ctccctcaag ttagcctctg 6840 accatactgt aaatatgcaa gtacacacaa aactaatgaa aagccactca aaaaaagcaa 6900 © gcagggcctc taccgaggec gaggcaaaga aggaatgacc taaattctce cgectgcage 6960 tgaaggcagg actagtgact caggaaagca ggtttaggcc ctcctagttt tgggctttgg 7020 ggtttcctgg attccctgac tgactcttcc ctgetgtgtc ttgaacctcc ttcaggecccg 7080 tattactgcg gtgtgggagc agacaaggcc tacggcaggg acatcgtgga ggctcactac 7140 cgggcctget tgtatgctgg agtcaagatt acggggacaa atgcggaggt tatgcctgec 7200 caggtaaatg gtgcccatct ttttcctcct ctctgaagac ctgggtaggt aggcacatgg 7260 ggacttcggg ctagcagggg tggatcacaa agtggggcaa tcacagaggg tggatcttaa 7320 aggtcaactt tttctctcta gtgggaattc cagataggac cctgtgaggg gatccgaatg 7380 ggagatcatc tttggatagc ccgttttatc ttgcatcggg tgtgcgaaga ctttggggtg 7440 atagcaacct ttgaccccaa gcccattcca gggaactgga atggtgcagg ctgccatacc 7500 aacttcagca ccaaggccat gcgggaggag aatggtciga agtaagtacc ttcctttgga 7560 gccgtctgta ttctcatggg gtagaagggc tttgggtact cacaaggctg tacgtacatg 7620 cctagctctg catatttgtt ctaagcctgt cagtttgtgc ctgttggaga ggcgataggg 7680 taaatacttt aggaatagaa ttgacagaaa agcattcgaa ctaagtaaaa atacaagcaa 7740

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S1546PCT25.5T25. txt atgggaaact taattcttac tggtgggaag aggcgagtga ttgggggtct ttccatccag 7800 . tggataattt gcactgcatg ttaaagactg gcctgaggga gacagtgcct tctttcttct 7860 gggattcatg cccgctccca tccttgtcga tggaccctca tecttcactgt ttccactagg 7920 tgcattgagg aggccattga caaactgagc aagaggcacc agtaccacat tcgcgcctac 7980 gatcccaagg ggggcctgga caacgcccgg cgtctgactg gattccacga aacctccaac 8040 atcaacgact tttctgccgg tgttgccaac cgcggtgcca gtatccgcat tccccggact 8100 gtcggccagg agaagaaggg ctactttgaa gaccgtcgge cttctgccaa ttgtgacccc 8160 tatgcggtga cagaagccat cgtccgcacg tgtctcctca acgaaacagg cgacgaaccc 8220 ttccaataca agaactaagt ggactagact tccagtgatc cctctcccag ctctteectt 8280 tcccagttgt ccccactgta actcaaaagg atggaatacc aaggtctttt tattcctcgt 8340 gcccagttaa tcttgettitt gttggtcaga atagaggggt caggttctta atctctacac 8400 accaaccctt tctttcctat ctagctttct agtagggagc gggagggggg aggggaaggg 8460 taacccactg cttcatctca tcgggtatge atgtccggta ggcatagctg tcacaaagcg 8520 ggtgtactta tggtgaaaga ggacattttt tttttcttca ggatagttga aagggcaggc 8580 ccaacggctg agattgacat ttccactgtt ggtagagagc tgttatttct aaaggggaaa 8640 ccagctttct gttccaaatg gaagttaggt gaggagttga aggttggttt cttgcgctgt 8700 gcttccttgg cttgggggag ggggcatccg tccccctctg tgtgaacaca gctcaccgeg 8760 tcacctgatg gatggcccta ctgtgaagga agaaaaaagt tggcatttct tggtcctccg 8820 ttcataacac aaagcagagt agtattttta tatttaaatg ttaaaaacaa aaaagttata 8880 ' tatatatgga tgtgtggatg tatgtctttc taattgagag aaccatccta ttcactgggt 8940 gccaagtttg agtgatgagg ggcttggctt agaagtgagg ctcccttgag gtaggggtga 9000 ggatgcagta ccgggaaagt tggttatctt ggggtctcag cttcattact gcttagggtt 9060 tccctgecca ctctgcagga gcagatgttg gacaggtagc cagtgggatg ccactgettg 9120 ccaccacctg tccccaggct taggtttagg ggatgcgtat acttactcca cacacgagtt 9180 agaagtatga gttggctggt caacttgaac actgttactg atgggtgggt gggtgggggt 9240 ttactggggt tatttttttg gtgggattag catgtcacta aagcgggect tttgatatat 9300 taagtttttt aaaagcaaaa caagtttaga ttttaatcag atttgtaggg tttctaactt 9360 tacagaattg cctgtttgtt tcaatgtctc cctccacttg gctcttaggg gaattaagga 9420 caggcctaga gttaaaacac ttgtctccta gtgtcacctc tgccagcaga ctgttacttt 9480 ccttctgaaa aagccaatag tctttttttt tttcttttat agtaaacaca cccccacctc 9540 catcccagce tgttgccctt cagttttctg gttgtttgtg tcggcagcgg gccaactgtg 9600 gtttctctct tgccatgatg acttctaatt gccatgtata gtatgttcgg ttagataact 9660 cactgtaaac agactgtaac tgccaggcag cgcttataaa tcaacctaac atttataaga 9720 tttcctctga cttgtttctt tgtggttccc aaaaaaaaaa aaaaaacctc 9770

Page 6 s1546PCT25.5T25.tXt <210> 4 <211> 167 <212> PRT <213> artificial sequence <220> <223> ICreI N75 scaffold protein <400> 4

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 . val Gly Tyr val Arg Asp Arg Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 5 <211> 24 <212> DNA . <213> artificial sequence <220> Co <223> human Glutamine Synthetase gene target <400> 5 ccatgaccac ctcagcaagt tccc 24

Page 7 s1546PCT25.5T25. txt <210> 6 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 6 catgtatatc tggatcgatg gtac 24 <210> 7 <211> 24 i ; ’ <212> DNA <213> artificial sequence . <220> <223> human Glutamine Synthetase gene target <400> 7 tgtatatctg gatcgatggt actg 24 <210> 8 <211> 24 <212> DNA <213> artificial sequence <220> } <223> human Glutamine Synthetase gene target <400> 8 tctggatcga tggtactgga gaag 24 <210> 9 <21l> 24 : <212> DNA <213> artificial sequence i <220> <223> human Glutamine Synthetase gene target <400> 9 ccggaccctg gacagtgagc ccaa 24 <210> 10 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 10 cctggacagt gagcccaagt gtgt 24 <210> 11 <21ll> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target

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§1546PCT25.5T25. txt <400> 11 aggaccctaa caagctggtg ttat 24 <210>- 12 <211> 24 <212> DNA <213> artificial sequence <220> } <223> human Glutamine Synthetase gene target . <400> 12 tataccctca tggggacaga tggg 24 <210> 13 <211> 24 <212> DNA <213>. artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 13 tgggcaccce tttggttgge cttc 24 <210> 14 <21}> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 14 gcttcccagg geccccagggt aagt 24 <210> 15 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 15 tcctgatget tctgtaggtc cata 24 <210> 16 <211> 24 . <212> DNA . <213> artificial sequence : <220> . <223> human Glutamine Synthetase gene target <400> 16 cggggactaa tgccgaggtc atgc . 24 <«210> 17 : <211> 24 <212> DNA <213> artificial sequence

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51546PCT25.5T25. txt <220> <223> human Glutamine Synthetase gene target <400> 17 tcatgcctge ccaggtaagt atag 24 <210> 18 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 18 agcaaccttt gatcctaagc ccat 24 <210> 19 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 19 caaggccatg cgggaggaga atgyg 24 <210> 20 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 20 cttttctgtt tactctaggt acat 24 <210> 21 <211> 24 <212> DNA . <213> artificial sequence <220> ] <223> human Glutamine Synthetase gene target <400> 21 : gtaccacatc cgtgcctatg atcc 24 <210> 22 <211> 24 . <212> DNA <213> artificial sequence <220> . <223> human Glutamine Synthetase gene target <400> 22 ccgtgectat gatcccaaqgg gagg 24

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51546PCT25.5T25,. txt <210> 23 <211> 24 <212> DNA . <213> artificial sequence <220> . <223> human Glutamine Synthetase gene target <400> 23 cctggacaat gcccgacgte taac 24 <210> 24 - <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target <400> 24 gcccgacgtc taactggatt ccat 24 <210> 25 <211> 24 <212> DNA : <213> artificial sequence <220> . : <223> human Glutamine Synthetase gene target <400> 25 - ttctgctggt gtagccaatc gtag } 24 <210> 26 <211> 24 . : <212> DNA <213> artificial sequence <220> . <223> human Glutamine Synthetase gene target <400> 26 gcattccccg gactgttggc cagg 24 <210> 27 <211> 24 <212> DNA <213> artificial sequence <220> . <223> human Glutamine Synthetase gene target <400> 27 ccggactgtt ggccaggaga agaa 24 <210> 28 <211> 24 <212> DNA <213> artificial sequence <220> <223> human Glutamine Synthetase gene target

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51546PCT25.5T25. tXt <400> 28 gttggccagg agaagaaggg ttac 24 <210> 29 <211> 24 <212> DNA : <213> artificial sequence <220> - <223> mouse Glutamine Synthetase gene target <400> 29 catggccacc tcagcaagtt ccca 24 <210> 30 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 30 ctgccccagg gtgagaaagt ccaa 24 <210> 31 <21i> 24 . <212> DNA <213> artificial sequence : : <220> . <223> mouse Glutamine Synthetase gene target <400> 31 tgtatatctg ggttgatggt accy 24 <210> 32 <21i> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 32 ccgtaccctg gactgtgagc ccaa 24 <210> 33 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 33

Cggaaacctyg caggcaagta tggg 24 <210> 34 <211> 24 <212> DNA <213> artificial sequence .

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I — s1546PCT25.5T25. txt <220> <223> mouse Glutamine Synthetase gene target <400> 34 agcaaccagc acccctggtt tgga 24 <210> 35 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 35 ctcttatggg aacagacggc cacc 24 <210> 36 <211> 24 <212> DNA <213> artificial sequence <220> . <223> mouse Glutamine Synthetase gene target <400> 36 gcttccctgg accccaaggt acgt 24 <210> 37 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 37 cggggacaaa tgcggaggtt atgc 24 <210> 38 <211> 24 <212> DNA <213> artificial sequence <220> . <223> mouse Glutamine Synthetase gene target <400> 38 gcctgeccag gtaaatggtg ccca 24 <210> 39 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 39 gtcaactttt tctctctagt ggga 24 : Page 13 s1546PCT25.5T25. txt <210> 40 <211> 24 ~ <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 40 ' taggaccctg tgaggggatc cgaa 24 <210> 41 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 41 tttggatagc ccgttttatc ttgc 24 <210> 42 <211> 24 <212> DNA <213> -artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 42 ’ gttttatctt gcatcgggtg tgcg 24 <210> 43 <211l> 24 ‘ <212> DNA <213> artificial sequence <220> <223> mouse Glutamine synthetase gene target <400> 43 tttatcttgc atcgggtgtg cgaa 24 <210> 44 <211> 24 <212> DNA <213> artificial sequence <220> ] <223> mouse Glutamine Synthetase gene target <400> 44 tcgegectac gatcccaagg gggg 24 <210> 45 <211> 24 <212> DNA <213> artificial sequence <220> . <223> mouse Glutamine Synthetase gene target

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51546PCT25.5T25. txt <400> 45 cctggacaac gcccggcgtc tgac 24 <210> 46 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 46 gcattccccyg gactgtcgge cagg 24 <210> 47 <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target . <400> 47 ccggactgtc ggccaggaga agaa 24 <210> 48 ’ <211> 24 <212> DNA <213> artificial sequence <220> <223> mouse Glutamine Synthetase gene target <400> 48 gtcggccagg agaagaaggg ctac 24 <210> 49 <211> 24 <212> DNA <213> artificial sequence <220> . <223> Chinese Hamster Glutamine Synthetase gene target <400> 49 tgtatatctg ggttgatggt actg 24 <210> 50 <211> 24 <212> DNA <213> artificial sequence <220> . <223> chinese Hamster Glutamine Synthetase gene target <400> 50 ccgcaccctyg gactgtgage ccaa 24 <210> 51 <211> 24 <212> DNA <213> artificial sequence

Page 15

51546PCT25.5T25. txt <220> <223> chinese Hamster Glutamine Synthetase gene target <400> 51 ctcagccctg ttgccatgtt tcgg 24 <210> 52 <211> 24 <212> DNA <213> artificial sequence <220> CL <223> chinese Hamster Glutamine Synthetase gene target <400> 52 cgggacccct tccgcagaga tccc 24 <210> 53 <211> 24 <212> DNA <213> artificial sequence <220> <223> chinese Hamster Glutamine Synthetase gene target <400> 53 tgggcaccct tttggttggc cttc 24 ’ <210> 54 <211> 24 <212> DNA <213> artificial sequence <220> Co <223> chinese Hamster Glutamine Synthetase gene target <400> 54 caaagcctat ggcagggata tcgt 24 <210> 55 <211> 24 <212> DNA <213> artificial sequence <220> <223> chinese Hamster Glutamine Synthetase gene target <400> 55 caggaacaaa tgctgaggtc atgc 24 <210> 56 <211> 24 <212> DNA <213> artificial sequence <220> } <223> chinese Hamster Glutamine Synthetase gene target <400> 56 ttcatcttgc atcgagtatg tgaa 24 page 16 s1546PCT25.5T25. txt <210> 57 <211> 24 <212> DNA <213> artificial sequence <220> <223> Chinese Hamster Glutamine Synthetase gene target '<400> 57 tcgagcctac gatcccaagg ggdg 24 <210> 58 <211> 24 <212> DNA <213> artificial sequence <220> : <223> Chinese Hamster Glutamine Synthetase gene target <400> 58 . cctggacaat gcccgtggtc tgac 24 <210> 59 <211> 24 <212> DNA <213> artificial sequence <220> <223> Chinese Hamster Glutamine Synthetase gene target <400> 59 ttctgcetggt gtcgccaatc gcag 24 <210> 60 <211> 24 <212> DNA : <213> artificial sequence <220> . <223> Chinese Hamster Glutamine Synthetase gene target <400> 60 gtcggccagg agaagaaagg ttac 24 <210> 61 <211> 163 <212> PRT <213> artificial sequence <220> . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 61

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln

Ser Gly Phe Lys His Gln Leu Ser Leu Thr phe val Thr Gln Lys Thr

Page 17

$1546PCT25.5T25. txt 35 40 45

GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly val Gly 50 55 60 :

Tyr val Glu Arg Gly Ser val Ser Asn Tyr Arg Ser Lys Ile Lys Pro 65 70 75 BO

Leu His Asn Phe Leu Thr 61n Leu Gln Pro Phe Leu Lys Leu Lys Gln 85 90 95

Lys .GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln Leu Pro Ser Ala 100 105 110

Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr Trp val Asp Gln 115 120 125

Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr ser Glu Thr . 130 135 140 val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys Lys Ser Ser Pro 145 150 155 160

Ala Ala Asp <210> 62 . <211> 167 <212> PRT <213> artificial sequence <220> . . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 62

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Gly Cys Lys Phe Lys His Ala Leu Ser Leu Thr phe Ile val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asn Tyr Ser Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Page 18 s15346PCT25.5T25. txt

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp G1ln Ile Ala Ala Leu Ash Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 63 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 63

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gin Ile Lys Pro Arg Gln

Thr Cys Lys Phe Lys His Gln Leu Thr Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr val Leu Ser 65 70 75 80

GTu Ile Lys Pro Leu His Asn Phe Leu Thr Gn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile ITe Glu Gln ‘ 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp G1n Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 . 135 140

Page 19

S1546PCT25.ST25. txt Co

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 :

Lys Ser Ser Pro Ala Ala Asp 165 <210> 64 <211l> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 64

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln ser Cys Lys Phe Lys His Ala Leu Ser Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 .. 60 : val Gly Tyr val Thr Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp G1n Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 i <210> 65 <211> 167 <212> PRT <213> artificial sequence

Page 20

L s1546PCT25.5T25. txt <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 65

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln ser Arg Lys Phe Lys His Asn Leu Gln Leu Thr phe Arg val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr : 115 120 125

Trp val asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 66 <21l> 167 <212> PRT <213> artificial sequence <220> . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 66

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val asp Gly Asp Gly Ser Ile Ile Ala Gin Ile Lys Pro Asn Gln 20 25 30 ’

Page 21 s1546PCT25.5T25. txt .

Thr Cys Lys Phe Lys His GIn Leu Ser Leu Thr Phe Leu val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ala Gly Ser val Ser Asn Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

BS 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val teu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe teu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala teu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 67 <211> 167 <212> PRT <213> artificial sequence <220> . <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 67

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

His His Lys Phe Lys His Gln Leu Ser Leu Thr phe Asn val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys teu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Gln Asp Ser Gly Ser val Ser Ser Tyr Val Leu Ser 65 70 75 BO

Page 22 s1546PCT25.5T25. txt

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 }

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp . 165 <210> 68 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 68

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Cys Cys Lys Phe Lys His Gln Leu Glu Leu Thr Phe Lys val Thr GIn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Thr Asp Thr Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 . 120 125

Page 23

—_— : s1546PCT25.5T25. txt

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155% 160

Lys Ser Ser Pro Ala Ala Asp 165 i <210> 69 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 69 ’

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Ser Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Arg Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

B5 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 70

Page 24 s1546PCT25.5T25. txt . <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 70

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln ser Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Arg Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 0 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 ‘

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 .

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 71 <211> 167 <212> PRT <213> artificial sequence <220> . . . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 71

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Page 25 s1546PCT25.5T25. txt

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gln ser Arg Lys Phe Lys His Thr Leu Ser Leu Thr Phe Arg val Thr Gln : 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asn Tyr Asp Leu Ser 65 70 75 : 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 35 90 | 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 i <210> 72 <211> 167 : <212> PRT. <213> artificial sequence <220> . . . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 72

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln 20 25 30

His His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Tyr val Thr Gln 35 4Q 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Page 26

51546PCT25.5T25.tXt val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Thr Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu G1n Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 73 . <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving. human Glutamine Synthetase gene target <400> 73

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln ser Gly Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

BS 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr

Page 27 s1546PCT25.5T25. txt i 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Led Ser Glu Lys Lys ‘ 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 74 <211> 167 <212> PRT i <213> artificial sequence <220> . <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 74 , Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 : 10 15 . Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln } 25 30

Ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln "35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr GIn Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 } Page 28 s1546PCT25.5T25. txt . <210> 75 <211> 167 : <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 75 .

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln

Ser His Lys Phe Lys His Lys Leu Ser Leu Thr phe Ile val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 76 <211> 167 <212> PRT } <213> artificial sequence <220> . . . <223> First I-Crel variant cleaving human Glutamine Synthetase gene target : <400> 76

Page 29 s1546PCT25.5T25. txt

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly } 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln ser Thr Lys Phe Lys His GIn Leu Thr Leu Thr phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ser Gly Ser val Ser Gln Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gin Leu GIn Pro Phe Leu 85 90 . 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 77 . <211> 167 <212> PRT <213> artificial sequence <220> . <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 77

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30 ser Arg Lys Phe Lys His Asp Leu Arg Leu Thr Phe Ile val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly

Page 30

S1546PCT25.5T25. txt 50 55 60 val Gly Tyr val Arg Asp Cys Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys G1In Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 78 <211> 167 <212> PRT __ <213> artificial sequence <220> . <223> First I-CreX variant cleaving human Glutamine Synthetase gene target <400> 78

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln .

Ser Pro Lys Phe Lys His Lys Leu Ser Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu 1le Gly 50 55 60 val Gly Tyr val Lys Asp His Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu &ln 100 105 110

Page 31 s1546PCT25.5T25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr . 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 .

Lys Ser Ser Pro Ala Ala Asp 165 <210> 79 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 79

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln : ~ 30

Ser Cys Lys Phe Lys His Ala Leu Ser Leu Thr phe Ile val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ala Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro teu His Asn Phe Leu Thr Gln teu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 : 120 125

Trp val Asp GIn Ile Ala Ala teu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Page 32

5154B6PCT25.5T25. txt

Lys Ser Ser Pro Ala Ala Asp 165 <210> 80 i <211> 167 <212> PRT . <213> artificial sequence <220> <223> First I-CreI variant cleaving human Glutamine Synthetase gene target <400> 80

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn GIn

Thr Tyr Lys Phe Lys His Trp Leu Ser Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Leu Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val teu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 81 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target

Page 33 s1546PCT25.5T25. txt <400> 81

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asp GIn ser Arg Lys Phe Lys His Ser Leu Ser Leu Thr Phe Tyr val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val ser Asp Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

BS 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr : 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140 : Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 82 . <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI varjant cleaving human Glutamine Synthetase gene target <400> 82 '

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln 20 25 30 ser Gly Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys val Thr Gln 35 40 45

Page 34

$1546PCT25.5T25. txt

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asp Tyr Lys Leu Ser 65 70 75 80

Glu Tle Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pra Phe Leu 8S 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Giu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys © 145 150 155 160

Lys Ser ser Pro Ala Ala Asp 165 <210> 83 : <211> 167 : <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> 83

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Asn Leu GIn Leu Thr Phe ser val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Tyr Tyr Thr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95 : Page 35

51546PCT25.5T25. txt

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys® 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 84 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving human Glutamine Synthetase gene target <400> B84

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly i 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln : .

Ser Cys Lys Phe Lys His Ala Leu Ser Leu Thr Phe Ala val Thr GlIn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 75 80

Glu Ile LYS Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Page 36 s1546PCT25.5T25. txt

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 85 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 85

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Thr Asn Lys Phe Lys His Gln Leu Gln Leu Thr Phe Asp val Thr Gn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly ser val ser Arg Tyr Thr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 a5

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln : 100 - 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 86 <211> 167 <212> PRT <213> artificial sequence

Page 37 s1546PCT25.5T25. txt <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 86

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln ser Arg Lys Phe Lys His Asp Leu Arg Leu Thr Phe Tyr val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Ser Gly Ser val Ser Arg Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 S90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr ‘ 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 87 <211> 167 <212> PRT <213> artificial sequence <220> ) . <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 87

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg GIn 20 25 30 . Page 38 s1546PCT25.5T25. txt

Thr Asn Lys Phe Lys His GIn Leu Gln Leu Thr Phe Leu val Thr Gln 35 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Asn Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Lys Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 20 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr : 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 88 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 88

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Ser Thr Lys Phe Lys His Ala Leu Ser Leu Thr Phe Met val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Arg Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Page 39

S1546PCT25.5T25. txt

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95 :

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln : 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 89 <211> 167 <212> PRT <213> artificial sequence <220> . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 89

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Glu Pro Asn Gln : 30

Ser Arg Lys Phe Lys His Arg Leu Lys Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr

Page 40 .

— s1546PCT25.5T25. txt 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 90 <211> 167 <212> PRT <213> artificial sequence <220> . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target : <400> 90

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Tyr GIn

Thr Cys Lys Phe Lys His Gln Leu Ser Leu Thr phe Asp val Thr Glin 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 91 <211> 167

Page 41

= — s1546PCT25.5T25. TXT <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 91

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Glu Lys Phe Lys His GIn Leu Glu Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Lys Leu Ser 65 70 75 80 :

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 92 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 92

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Page 42 s1546PCT25.5T25. txt

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn GIn

Arg Asp Lys Phe Lys His Glin Leu ser Leu Thr phe Ala val Thr Gln 40 45 ~ Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Leu Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 ’ 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 .

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 93 . <211> 167 <212> PRT . <213> artificial sequence <220> . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target i <400> 93

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln 20 25 30

Ser His Lys Phe Lys His Lys Leu Ser Leu Thr Phe Asn val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser

Page 43

51546PCT25.ST25. txt 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 as ,

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln : 100 105 110 :

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 .

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys . 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 94 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 94

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Ser Pro Lys Phe Lys His Gln Leu Gln Leu Thr Phe Lys val Thr Gln 40 4

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val ser Glu Tyr val Leu Ser b> 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 a0 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 . Page 44 s1546PCT25.5T25. txt

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 95 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 95

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ash Gln

Cys Cys Lys Phe Lys His His Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Ash Tyr Asp Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165

Page 45 s1546PCT25.ST25. txt <210> 96 <211l> 167 <212> PRT <213> artificial sequence <220> . . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 96

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln

Ser Gly Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Tyr Tyr Arg Leu Ser 65 70 75 : 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 . 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe teu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 97 <211> 167 <212> PRT <213> artificial sequence <220> } <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 97

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly

Page 46 s1546PCT25.5T25. txt 1 5 10 15 :

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Ala Leu GIn Leu Thr Phe Asn val Thr Gln 40 45 ‘

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 .

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 98 <211> 167 <212> PRT <213> artificial sequence <220> . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 98

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Glu Pro Asn GIn 20 25 30 ser Arg Lys Phe Lys His Arg Leu Lys Leu Thr Phe Lys val Thr Gln 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Page 47

§1546PCT25.5T25. txt val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Gln Tyr Asn Leu Ser 65 70 } 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 ;

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 : 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 99 <211> 167 <212> PRT <213> artificial sequence <220> . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target . <400> 99

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ash Gln

Asp Tyr Lys Phe Lys His Cys Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu : 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Page 48 s1546PCT25.5T25. txt . Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 :

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 100 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 100

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly ’ 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser arg Lys Phe Lys His Ala Leu G1n Leu Thr Phe Ala val Thr Gin 40 45

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Gln Asp Asn Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Tle Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Page 49

$1546PCT25.5T25. XT

Lys Ser Ser Pro Ala Ala Asp 165 <210> 101 <211> 167 <212> PRT <213> artificial seguence <220> } <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 101

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln :

Ala Asn Lys Phe Lys His Gln Leu Glu Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 a5

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 102 <211> 167 <212> PRT <213> artificial sequence <220> . . . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target page 50 s1546PCT25.5T25. txt . <400> 102

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Arg Pro Asn Gln :

Ser Ala Lys Phe Lys His Tyr Leu Gln Leu Thr phe Asp val Thr Gin . 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 103 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 103

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30

Thr Cys Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Page 51

$1546PCT25.5T25. txt

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser His Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln ’ 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 120 125

Trp val Asp Gln Ile Ala Ala Leu Ash Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 104 <211> 167 <212> PRT ’ <213> artificial sequence <220> ) . <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 104

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Asn Leu GIn Leu Thr Phe Tyr val Thr Glin 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 . val Gly Tyr val Ser Asp Ser Gly Ser val Ser Ser Tyr Asp Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Page 52

§1546PCT25.ST25. txt

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 .

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr i 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 "Lys Ser ser Pro Ala Ala Asp 165 <210> 105 <211l> 167 <212> PRT : <213> artificial sequence <220> <223> Second I-Crel variant cleaving human Glutamine Synthetase gene target <400> 105

Met Ala Asn Thr Lys Tyr Ash Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 rhe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Thr Tyr Lys Phe Lys His Trp Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 : 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe teu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala teu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys

Page 53 s1546PCT25.5T25. txt 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 106 <211> 167 <212> PRT . <213> artificial sequence <220> <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 106

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Asn Pro Asn Gln

Ser Ser Lys Phe Lys His Arg Leu Lys Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly : 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 107 <211> 167 : <212> PRT <213> artificial sequence <220>

Page 54 s1546PCT25.5T25. txt <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 107 :

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gln : 25 30

Ser Arg Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu : 85 80 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 108 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving human Glutamine Synthetase gene target <400> 108

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Page 55 s1546PCT25,5T25. txt .

Ser Arg Lys Phe Lys His GIn Leu Ser Leu Thr phe Gln val Thr Gin 35 40 45

Lys Thr ¢1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Tyr Asp Arg Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr &1n Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 : 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 © <210> 109 <211> 167 <212> PRT <213> artificial sequence <220> . : <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 109

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Cys Cys Lys Phe Lys His His Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr G1In Leu Gln Pro Phe Leu

Page 56 s1546PCT25.5T25. txt

BS 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 :

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp G1n Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 . <210> 110 <211> 167 <212> PRT . <213> artificial sequence <220> <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 110

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro phe Leu

BS 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp G1n Ile Ala Ala Leu Ash Asp Ser Lys Thr Arg Lys Thr 130 135 140 : . Page 57

§1546PCT25.5T25. txt

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 111 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target , <400> 111

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 ]

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro His Gln

His Cys Lys Phe Lys His Gln Leu Thr Leu Thr Phe Arg val Thr GIn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile 1le Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 112 <21l> 167 <212> PRT

Page 58 :

} s1546PCT25.5T25. txt <213> artificial sequence <220> , <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target ’ <400> 112

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Glin

Ser Arg Lys Phe Lys His Ala Leu GIn Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 - 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 113 <211> 167 <«212> PRT <?213> artificial sequence <220> . . <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 113

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Page 59

51546PCT25.5T25. txt

Gly Arg Lys Phe Lys His Gln Leu Ala Leu Thr Phe Arg val Thr Gn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 . 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 . 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 114 <211> 167 . <212> PRT <213> artificial sequence <220> <223> First I-CreIl variant cleaving mouse Glutamine Synthetase gene target ‘ <400> 114

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly . 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln 20 25 30 ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Leu val Thr GIn 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Asn Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Page 60 s1546PCT25.5T25. txt

Lys Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 a0 a5

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr } 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 115 . <211> 167 <212> PRT <213> artificial sequence <220> i : <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 115

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gin ser Arg Lys Phe Lys His Gin Leu Ser Leu Thr Phe Tyr val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asp Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn phe Leu Thr Gln Leu Gin Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Page 61 s1546PCT25.5T25. txt

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 116 <211> 167 <212> PRT <213> artificial sequence <220> } <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target "<400> 116

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Ser Thr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Asp val Thr Gln : 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp 165

Page 62 s1546PCT25.5T25. txt <210> 117 i <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 117 . Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln :

His His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly . 50 55 60 val Gly Tyr val His Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 118 <211> 167 : <212> PRT <213> artificial sequence <220> . . . <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 118 .

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Page 63 s1546PCT25.5T25. txt

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gln

Ser Arg Lys Phe Lys His Thr Leu Ser Leu Thr phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Lys Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 . 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 i <210> 119 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CrelI variant cleaving mouse Glutamine Synthetase gene target <400> 119

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn GIn 20 25 30

Ser Cys Lys Phe Lys His GIn Leu Gln Leu Thr phe Gln val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Page 64

$1546PCT25.5T25. txt val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gin Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys G1n Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 120 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 120

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Tyr Lys Phe Lys His Gin Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gin Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Ash Leu val Leu Lys Ile Ile Glu Glin 100 105 110

Page 65 s1546PCT25.5T25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 )

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 121 : 3 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 121

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln ser Cys Lys Phe Lys His Ala Leu Ser Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 : 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Ash Phe Leu Thr Gln Leu Gln Pro Phe Leu - 85 90 95

Lys Leu Lys Gln Lys GIn Ala Ash Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp oo

Page 66

§1546PCT25.5T25.txt 165 <210> 122 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 122 i

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro asn Gln

Ser Ser Lys Phe Lys His Arg Leu Asp Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 i 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 123 <211> 167 <212> PRT <213> artificial sequence <220> . . . <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 123

Page 67 s1546PCT25.5T25. txt

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 30 .

Thr Cys Lys Phe Lys His GIn Leu Ser Leu Thr Phe Glu val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Cys Asp Ser Gly Ser val ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser ser Pro Ala Ala Asp 165 <210> 124 <211> 167 <212> PRT <213> artificial sequence <220> . . i i <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 124

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 : 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln 20 25 30

Ser His Lys Phe Lys His Arg Leu Ser Leu Thr Phe Asn val Thr Gln 35 40 45

Page 68

$1546PCT25.5T25. txt ’

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser ser Pro Ala Ala Asp 165 <210> 125 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving mouse Glutamine Synthetase gene target <d400> 125

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Tle Ile Ala Gln Ile Lys Pro Ser Glin : ser Ser Lys Phe Lys His His Leu Ser Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu GIn

Page 69 s1546PCT25.5T25. txt 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 126 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 126

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly i 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp GIn ser Arg Lys phe Lys His Gln Leu Ser Leu Thr phe Lys val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 20 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys : 145 150 155 160

Page 70

$1546PCT25.5T25. txt

Lys Ser Ser Pro Ala Ala Asp 165 . <210> 127 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-crel variant cleaving mouse Glutamine Synthetase gene target <400> 127

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 }

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Asn Leu Gln Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Leu Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu : 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp 165 : <210> 128 <211> 167 <212> PRT <213> artificial sequence <220> . <223> First I-Crel variant cleaving mouse Glutamine Synthetase gene

Page 71 s1546PCT25.5T25. txt target <400> 128

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 .

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Gly Gln

Ser Tyr Lys Phe Lys His Lys Leu Ser Leu Thr phe Thr val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly ser val ser Glu Tyr Arg Leu Ser 65 70 75 80 :

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 129 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene / target : <400> 129

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Ser Tyr Lys Phe Lys His Gln Leu Ser Leu Thr phe Thr val Thr Gln

Page 72 s1546PCT25,5T25. txt 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Thr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 a0 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 : - Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 : 150 155 160

Lys Ser Ser pro Ala Ala Asp 165 <210> 130 <211> 167 <212> PRT <213> artificial sequence <220> } ) } <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 130

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr phe arg val Thr GIn 40 45 . Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 . 90 95

Page 73

§1546PCT25.5T25. txt

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 .

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp ’ 165 <210> 131 <211> 167 . <212> PRT <213> artificial sequence <220> . } <223> second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 131

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20° 25 30

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 . 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu

B85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 }

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Page 74

51546PCT25.5T25. txt

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 ]

Lys Ser Ser Pro Ala Ala Asp 165 <210> 132 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 132

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser £5 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 .

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 : 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 133 <211> 167 <212> PRT <213> artificial sequence

Page 75 s1546PCT25.5T25. txt <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 133

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Cys Tyr Lys Phe Lys His Gln Leu Ser Leu Thr rhe Arg val Thr Gln i 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 134 <211> 167 <212> PRT <213> artificial sequence : <220> . . <223> second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 134

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln 20 25 30

Page 76 .

s1546PCT25.5T25. txt

Thr Asn Lys Phe Lys His Gln Leu GIn Leu Thr Phe Ile val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Cys Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 a0 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 150

Lys Ser Ser Pro Ala Ala Asp 165 <210> 135 <211> 167 <212> PRT <213> artificial sequence <220> . . . <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 135

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Ser His Lys Phe Lys His Ala Leu Ser Leu Thr phe val val Thr Gln - 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Arg Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Page 77 s1546PCT25.5T25. txt

Lys Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr . 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160 .

Lys Ser Ser Pro Ala Ala Asp . 165 <210> 136 <211> 167 <212> PRT <213> artificial sequence oe <220> . . <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 136

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Tle Lys Pro Asn GIn

Arg Asp Lys Phe Lys His GIn Leu Ser Leu Thr Phe Thr val Thr G1n 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 35 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Thr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

B5 30 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Page 78 s1546PCT25.5T25. txt

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 . 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 137 <211l> 167 <212> PRT <213> artificial sequence } <220> : <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 137

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile val Ala Gln Ile Lys Pro Asn Gln ser Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Asn Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu

BS 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 ,

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 138 : Page 79 s1546PCT25.5T25. txt <211> 167 <212> PRT . <213> artificial sequence <220> } <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 138

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro His GIn

His Cys Lys Phe Lys His Ala Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 30 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Gln Leu Ser 635 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 .

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys LyS 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 139 <211l> 167 <212> PRT <213> artificial sequence <220> . . <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 139

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Page 80 s1546PCT25.5T25. txt

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Ala Leu GIn Leu Thr phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ser Gly Ser val Ser Gln Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 50 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 140 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 140

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30 ser Gly Lys Phe Lys His GIn Leu GIn Leu Thr Phe GIn val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Page 81 s1546PCT25.5T25. txt val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Val Leu Ser 65 70 75 BO :

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 i

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 ’ 155 160

Lys Ser Ser Pro Ala Ala ASp 165 <210> 141 <21l> 167 <212> PRT _ <213> artificial sequence <220> : } <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 141

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Thr Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ser Asp Ser Gly Ser val Ser Asn Tyr Thr Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr

Page 82 s1546PCT25.5T25. txt 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 142 <211> 167 <212> PRT . <213> artificial sequence <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 142

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Asp Tyr Lys Phe Lys His Tyr Leu Ser Leu Thr Phe Thr val Thr Gln .35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr-val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Glin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp 165

Page B3 s1546PCT25.5T25. txt <210> 143 <211> 167 <212> PRT <213> artificial sequence <220> <223> second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 143

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln ser Tyr Lys Phe Lys His Arg Leu Lys Leu Thr Phe Lys val Thr Gln 40 45 tys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Ser Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser . i 65 70 75 80 ; Glu ITe Lys Pro Leu His Asn Phe Leu Thr Gln teu GIn Pro Phe Leu 85 90 a5

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 144 : <211> 167 ' <212> PRT <213> artificial sequence <220> } ) . <223> Second I-CreI variant cleaving mouse Glutamine Synthetase gene target <400> 144

Page 84 s1546PCT25.5725. txt

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln i

Thr Tyr Lys Phe Lys His GIn Leu Ser Leu Thr phe Gln val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Ser Gly Ser val Ser Ash Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

B5 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 teu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe teu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys : 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 145 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 145

Met ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30

Ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr phe Lys val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly

Page 85

51546PCT25.5T25. txt 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80 - Glu ITe Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gin Pro Phe Leu

BS 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp G1In Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp 165 <210> 146 <211l> 167 <212> PRT . <213> artificial sequence, <220> . <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 146

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Asn Gly Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Glin 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly i 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Page 86

$1546PCT25.5T25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 :

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 147 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 147

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu .Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln : :

Thr Tyr Lys Phe Lys His Trp Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gin Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Page 87 s1546PCT25.5T25. Xt

LYS Ser Ser Pro Ala Ala Asp 165 <210> 148 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target . <400> 148

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 rhe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asp Gln

Ser Arg Lys Phe Lys His Gly Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser 65 70 75 : 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 :

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 149 <211> 167 <212> PRT _ <213> artificial sequence <220> <223> Second I-CrelI variant cleaving mouse Glutamine Synthetase gene target

Page 88 s1546PCT25.5T25. txt <400> 149

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gin Ile Asn Pro Asn Gin 30 .

Ser Tyr Lys Phe Lys His Arg Leu Lys Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Arg Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gin Leu Gln Pro Phe Leu 85 20 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 150 <211> 167 <212> PRT <213> artificial sequence <220> } . i <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 150

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Trp Gln 20 25 30

Ser Cys Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys val Thr Gln 35 40 45

Page 89 s1546PCT25.5T25. txt

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly ’ 50 55 60 val Gly Tyr val Ala Asp Ser Gly Ser val Ser Asn Tyr Lys Leu Ser 65 70 75 80

Glu ITe Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 a0 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Ara Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 151 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-Crel variant cleaving mouse Glutamine Synthetase gene target <400> 151

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 - Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn GIn : 30 ser Tyr Lys Phe Lys His Gly Leu GIn Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Gln Leu Ser 65 70 75 + 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 a0 95 . page 90

. $1546PCT25.5T25. txt

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln : 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr - 115 120 125

Trp Val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 152 : <211> 167 <212> PRT : <213> artificial sequence <220> <223> First I-CreI variant cleaving Chinese Hamster Glutamine : Synthetase gene target <400> 152

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

His Cys Lys Phe Lys His GIn Leu Ala Leu Thr Phe Arg val Thr GIn 3s 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln . 100 105 110

Leu Pro Ser Ala Lys Glu ser Pro ASp Lys Phe Leu Glu val Cys Thr 115 120 125 :

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Page 91

: 51546PCT25.5T25. txt : .

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys ’ 145 150 155 160 ,

Lys Ser Ser Pro Ala Ala Asp 165 <210> 153 <211> 167 <212> PRT . <213> artificial sequence <220> } <223> First I-Crel variant cleaving chinese Hamster Glutamine

Synthetase gene target <400> 153

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser ITe Ile Ala Gln Ile Lys Pro Ash Gln ser Arg Lys Phe Lys His Asp Leu Arg Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 : 60 : val Gly Tyr val Tyr Asp Ser Gly Ser val ser Glu Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 .

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys } 145 150 155 160 !

Lys Ser ser Pro Ala Ala Asp 165 <210> 154 <211> 167 <212> PRT . <213> artificial sequence

Page 92

S1546PCT25.5T25. txt oo <220> <223> First I-CreI variant cleaving Chinese Hamster Glutamine } Synthetase gene target i <400> 154

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Ash Gln

Ser Asn Lys Phe Lys His Gln Leu Arg Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys. Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 155 <211> 167 <212> PRT <213> artificial sequence <220> i i } <223> First I-CreI variant cleaving chinese Hamster Glutamine

Synthetase gene target <400> 155

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Page 93 s1546PCT25.5T25. txt ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 . val Gly Tyr val Glu Asp Ser Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80 clu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

BS a0 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 156 <211> 167 <212> PRT : <213> artificial sequence <220> , <223> First I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 156 }

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr Gn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ser Asp Ser Gly Ser val Ser Ash Tyr val Leu Ser 65 70 75 BO

Page 94

] s1546PCT25,ST25. txt

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 157 <211> 167 <212> PRT <213> artificial sequence <220> <223> First I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 157

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln

Ser Cys Lys Phe Lys His Gln Leu Ala Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Lys Asp His Gly Ser val Ser Asn Tyr Ile Leu Ser 65 : 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr

Page 95 s1546PCT25.5T25. txt 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser ser Pro Ala Ala Asp 165 <210> 158 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> First I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 158

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 . 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg GIn

Ser Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr G1ln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 159 <211> 167

Page 96 s1546PCT25.5T25. txt <212> PRT <213> artificial sequence <220> <223> First I-CreI variant cleaving Chinese Hamster Glutamine synthetase gene target <400> 159

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln

Arg Asp Lys Phe Lys His GIn Leu Ser Leu Thr Phe Glu val Thr GIn 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Cys Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 160 <211> 167 <212> PRT _ <213> artificial sequence <220> a a - 0 - . <223> First I-Crel variant cleaving Chinese Hamster Glutamine synthetase gene target <400> 160

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 ‘

Page 97 s1546PCT25.5T25. txt

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser His Lys Phe Lys His Gln Leu Thr Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 . val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95 :

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ald val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 161 <211> 167 <212> PRT : <213> artificial sequence <220> . . . . <223> First I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 161

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Asn Gln 20 25 30

Ser Cys Lys Phe Lys His His Leu Ser Leu Thr Phe val val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Arg Gly Ser val Ser Asn Tyr Arg Leu Ser

Page 98

$1546PCT25.5T25. txt 65 70 75 80

Lys Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Ir 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Ash Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 162 <211> 167 i <212> PRT <213> artificial sequence <220> i <223> First I-Crel variant cleaving chinese Hamster Glutamine

Synthetase gene target <400> 162

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gln

Ser Arg Lys Phe Lys His Thr Leu Ser Leu Thr Phe Tyr val Thr Gln 40 45 :

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asp Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Page 99 s1546PCT25.5T25. txt

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 163 <211> 167 <212> PRT i <213> artificial sequence <220> . . . <223> First I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 163

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Gly Gln

Ser Tyr Lys Phe Lys His Lys Leu Ser Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Glu Tyr Arg Leu Ser 65 70 73 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165

Page 100 s1546PCT25.5T25. txt <210> 164 . <211> 167 <212> PRT <213> artificial sequence <220> <223> second I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 164

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Thr Asn Lys Phe Lys His Gln Leu G1n Leu Thr phe Ile val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Tle Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 165 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> Second I-CreI variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 165 met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly

Page 101

. s1546PCT25.5T25. txt 1 5 10 . 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln © 30 Co ser Gly Lys Phe Lys His Gln Leu Gly Leu Thr Phe Thr val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu G1ln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu G1n 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 } 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 166 . <21l> 167 <212> PRT <213> artificial sequence <220> . . . <223> second I-CreI variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 166

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Asp Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Asn val Thr Gln 35 40 45 ’

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Page 102 s1546PCT25.5T25. txt val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser So 65 70 75 80 .

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 167 <211> 167 <212> PRT - <213> artificial sequence <220> : <223> Second I-Crel variant cleaving Chinese Hamster Glutamine synthetase gene target <400> 167

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ash Gln

His His Lys Phe Lys His GIn Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Glu Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 a5

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Page 103

. s1546PCT25.5T25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp Val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 168 <211> 167 <212> PRT <213> artificial sequence <220> . . <223> Second I-Crel variant cleaving Chinese Hamster Glutamine synthetase gene target <400> 168

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 : 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln

Thr Ser Lys Phe Lys His Arg Leu Ser Leu Thr Phe Ala val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser His Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 20 : 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Page 104 s1546PCT25.5T25. txt

Lys Ser Ser Pro Ala Ala Asp i 165 } ‘ <210> 169 <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 169

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Gly His Lys Phe Lys His GIn Leu Ser Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Glu Asp Ser Gly Ser val Ser Arg Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 0 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 170 <211> 167 <212> PRT _ <213> artificial sequence <220> . . . <223> Second I-CreI variant cleaving Chinese Hamster Glutamine

Synthetase gene target

Page 105

51546PCT25.5T25. txt <400> 170

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln

Ser Gly Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 a0 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp ‘ 165 <210> 171 <211> 167 <212> PRT <213> artificial sequence <220> . <223> Second I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 171

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30

Ser Asn Lys Phe Lys His Tyr Leu Arg Leu Thr Phe Gln val Thr Gln 35 40 45

Page 106 s1546PCT25.5T25. txt

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Asp Gly Ser val Ser Asn Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr G1n Leu Gln Pro Phe Leu

BS 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu G1In 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 i

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 172 <211> 167 <212> PRT <213> artificial sequence <220> ; <223> Second I-CreI variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 172 met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Tyr Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln ' 40 45 : Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly ! 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Page 107 s1546PCT25.5T25. txt

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu GJu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 173 } <211> 167 <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving Chinese Hamster Glutamine synthetase gene target <400> 173

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Ser Pro Asn GIn

Ser Tyr Lys Phe Lys His Arg Leu Lys Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gin Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys

Page 108 s1546PCT25.5T25. txt 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 174 <211l> 167 : <212> PRT <213> artificial sequence <220> <223> Second I-CreI variant cleaving chinese Hamster Glutamine synthetase gene target <400> 174

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Lys Gln

Ser Ser Lys Phe Lys His GIn Leu Ser Leu Thr Phe Asp val Thr Gln 40. 45

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ser Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn ‘ 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 175 <211> 167 <212> PRT <213> artificial sequence <220>

Page 109 s1546PCT25.5T25. txt <223> second I-Crel variant cleaving Chinese Hamster Glutamine

Synthetase gene target <400> 175

Met Ala Asn Thr Lys Tyr Asn LYS Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln

Ser Arg Lys Phe Lys His Gln Leu Arg Leu Thr phe Ala val Thr Gln 35 . 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Gly Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110°

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 176 <211> 24 <212> DNA <213> artificial sequence <220> <223> 10GCC_P target <400> 176 tcgeccacgtc gtacgacgtg gcga 24 <210> 177 : <21]1> 24 <212> DNA <213> artificial sequence <220> <223> 10GGA_P target

Page 110

.s1546PCT25.5T25. txt <400> 177 tcggaacgtc gtacgacgtt ccga 24 <210> 178 <211> 24 <212> DNA <213> artificial sequence <220> <223> S5AGG_P target <400> 178 tcaaaacagg gtaccctgtt ttga 24 <210> 179 . <211> 24 <212> DNA <213> artificial sequence <220> <223> 5TTC_P target <400> 179 tcaaaacttc gtacgaagtt ttga 24 <210> 180 <211> 22 : <212> DNA <213> artificial sequence <220> oo <223> GSCHOl.2 target ©. <400> 180 tgccccaggg tacgaaagtc ca 22 <210> 181 <211> 22 <212> DNA <213> artificial sequence <220> <223> GSCHOl.3 target <400> 181 tgccccaggg taccctgggg ca 22 <210> 182 <211> 22 <212> DNA ] <213> artificial sequence <220> <223> GSCHOl.4 target <400> 182 } tggactttcg tacgaaagtc ca 22 <210> 183 <211> 51 <212> DNA .

Page 111 oo $1546PCT25.5T25. txt <213> artificial sequence <220> <223> GSCHO1.3 oligonucleotide <400> 183 tggcatacaa gtttctgeccc cagggtaccc tggggcagca atcgtctgic a 51 <210> 184 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOL.3 variant of Table vI <400> 184

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 3 10 ) 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln 30 .

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Thr val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 :

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser ser Pro Ala Ala Asp 165 <210> 185 <211> 51 <212> DNA <213> artificial sequence

Page 112

51546PCT25.5T25. txt <220> : <223> GSCHOL1.4 oligonucleotide <400> 185 : tggcatacaa gtttttggac tttcgtacga aagtccaaca atcgtctgtc a 51 <210> 186 <211> 26 <212> DNA <213> artificial sequence <220> <223> GallQF primer <400> 186 gcaactttag tgctgacaca tacagg 26 <210> 187 <211l> 24 <212> DNA . <213> artificial sequence <220> <223> GallQr primer <400> 187 acaaccttga ttggagactt gacc 24 <210> 188 . <211> 15 <212> DNA <213> artificial sequence <220> : <223> assF primer . <220> <221> misc_feature <222> (4)..(6) . <223> nis a, c, g, ort <400> 188 ctannnttga ccttt 15 <210> 189 <211> 15 <212> DNA . <213> artificial sequence <220> - <223> assR primer : <220> <221> misc_feature : <222> (10)..(12) <223> nisa, ¢, g, ort <400> 189 aaaggtcaan nntag ’ 15 <210> 190

Page 113

51546PCT25.5T25. txt ] <211> 167 <212> PRT _ <213> artificial sequence <220> <223> GSCHOl.4 variant of Table VIII <400> 190

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ~ 230

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr Glin : 35 40 45 .

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80 i

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110 teu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 191 <211> 167 <212> PRT <213> artificial sequence <220> . <223> GSCHO1l.4 variant of Table VIII <400> 191

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln page 114 s1546PCT25,5T25. txt

Ser His Lys Phe Lys His Asn Leu Ser Leu Thr phe Lys val Thr Lys 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile ITe Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 192 <21l> 167 <212> PRT . . <213> artificial sequence <220> <223> GSCHOl1l.4 variant of Table VIII <400> 192

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly : 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr phe Lys val Thr Gln 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu Ala Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ala Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Page 115

51546PCT25.5T25. txt " Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu ‘ 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gin : 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 193 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOl.4 variant of Table VIII <400> 193

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Ser Thr Lys Phe Lys His Arg Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Asn Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu

BS 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr

Page 116

. 51546PCT25.5T25. txt 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 } <210> 194 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHO1.4 variant of Table VIII <400> 194

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Met Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Val Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr : 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 195 <211> 167 <212> PRT

Page 117

. S1546PCT25.5T25. Xt : <213> artificial sequence <220> : <223> GSCHOl.4 variant of Table VIII <400> 195

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg val Thr Glin 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser ; : 65 - 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95 :

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu G1n 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr : 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 196 : <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHO1.4 variant of Table VIII <400> 196 ;

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln 20 25 30

Page 118 s1546PCT25.5T25. txt

Ala His Lys Phe Lys His GIn Leu Ser Leu Thr Phe Lys val Thr GIn 35 : 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Pro Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 197 <211> 167 <212> PRT <213> artificial sequence <220> . <223> GSCHO1.4 variant of Table VIII <400> 197

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro His Gln

Arg His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr 61n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu G1n Pro Phe Leu

Page 119 s1546PCT25,5T25.txt 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 i

Lys Ser Ser Pro Ala Ala Asp 165 <210> 198 <211> 167 i <212> PRT i <213> artificial sequence <220> <223> GSCHOl.4 variant of Table VIII <400> 198

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn

Asp Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr In Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Thr Leu Ser 65 70 75 380

Lys Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro phe Leu : 85 30 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Page 120 s1546PCT25.5T25. txt

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys : 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 199 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOl.4 variant of Table VIII <400> 199

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn

Asp Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Thr val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Arg Asp Ser Gly Ser val Ser Arg Tyr Ile Leu Ser 65 70 75 80

Lys Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp ’ 165 <210> 200 <211> 167 <212> PRT <213> artificial sequence

Page 121 s1546PCT25.5T25. txt <220> <223> GSCHO1.4 variant of Table VIII , <400> 200

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 : 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr phe Gln val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly } 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 . <210> 201 <211> 51 <212> DNA <213> artificial sequence <220> <223> GSCHO01l.2 oligonucleotide <400> 201 tggcatacaa gtttctgccc cagggtacga aagtccaaca atcgtctgtc a 51 <210> 202 <211> 51 <212> DNA <213> artificial sequence <220> . <223> GSCHO1 oligonucleotide

Page 122

51546PCT25.5T25. txt <400> 202 tggcatacaa gtttctgccc cagggtgaga aagtccaaca atcgtctgtc a 51 <210> 203 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOl1.4 variant of Table X <400> 203

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 23 30

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 204 <211> 167 <212> PRT ’ <213> artificial sequence <220> <223> GSCHOl.4 variant of Table X <400> 204

Page 123

S1546PCT25.5T25. txt

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr phe Arg val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Thr Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 i <210> 205 <211l> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOLl.4 variant of Table X <400> 205 .

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Thr val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly

Page 124

S1546PCT25.5T25. txt 50 55 60 : val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr val Leu Ser oo : 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 206 <211> 167 <212> PRT .<213> artificial sequence - <220> <223> GSCHOl.4 variant of Table X <400> 206

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 - . 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Page 125 s1546PCT25.5T25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp . 165 <210> 207 <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHO1.4 variant of Table X <400> 207

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln : 20 25 : 30

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Asn val Thr Gln } 40 45

Lys Thr Gn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp

Page 126

5s1546PCT25.5T25. txt ’ 165 <210> 208 i <211> 167 <212> PRT <213> artificial sequence <220> <223> GSCHOl.4 variant of Table X <400> 208

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Lys Gln

Ser Ala Lys Phe Lys His GIn Leu Ser Leu Thr Phe Asn val Thr G1n 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Arg Tyr Tyr Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 20 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 : 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp } 165 <210> 209 <211> 59 <212> DNA <213> artificial sequence <220> . <223> preATGCreFor primer <400> 209 gcataaatta ctatacttct atagacacgc aaacacaaat acacagcggc cttgccacc 59

Page 127 s1546PCT25.5T25. txt <210> 210 <211> 48 <212> DNA . <213> artificial sequence ) <220> . <223> ICreIpostRev primer : <400> 210 ggctcgagga gctcgtctag aggatcgctc gagttatcag tcggecgc 48 <210> 211 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1l.3 variant of Table XII <400> 211

Met Ala Asn Thr Lys Tyr Asn Glu Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Ala Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln - 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr GIn Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Ala Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 212

Page 128 s1546PCT25.5T25. txt <211> 167 <212> PRT . <213> artificial sequence <220> <223> Optimized GSCHOl.3 variant of Table XII : <400> 212

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gin 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly His val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gin Leu Gin Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 :

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 213 <211> 167 <212> PRT <213> artificial sequence <220> CL . <223> Optimized GSCHOLl.3 variant of Table XII <400> 213 met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Page 129

51546PCT25.5T25. txt

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln i 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 , val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser . 65 70 75 80

Glu Ile Lys Pro Leu His Asn Leu Leu Thr Gln Leu Gln Pro Phe Leu : 85 a0 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 214 <211> 167 . <212> PRT <213> artificial sequence <220> LL <223> Optimized GSCHO1l.3 variant of Table XII <400> 214

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Leu Asp val Thr Gln 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Page 130

51546PCT25.5T25. txt

Glu Tle Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

BS q0 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 215 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1.3 variant of Table XII <400> 215 :

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp ser Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Glu Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu

B5 20 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Thr Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr

Page 131 s1546PCT25.5T25. txt : 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 216 <211> 167 <212> PRT <213> artificial sequence <220> <223> Optimized GSCHO1.3 variant of Table XII <400> 216

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile val Ala Gln Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu. Thr phe Thr val Thr Gln : 35 40 45

Lys Thr GTn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 ‘60 : val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Thr Leu Ser 65 70 75 80

Lys Ile Lys Pro Leu His Asn Phe Leu Thr G1n Leu Gln Pro Phe Leu 85 20 a5

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Arg Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys , 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp - 165 <210> 217 <211> 167 «212» PRT

Page 132 oo S1546PCT25.5T25. txt <213> artificial sequence <220> <223> optimized GSCHOLl.3 variant of Table XII <400> 217

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Ser arg Lys Phe Lys His Glu Leu Ser Leu Thr phe Thr val Thr Gln 40 45

Lys Thr Arg Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Ash Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

BS a0 as

Lys Leu Lys Gln Lys GIn Ala Ash Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 - 125 :

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 218 <211> 167 <212> PRT <213> artificial sequence <220> <223> Optimized GSCHOL.3 variant of Table XII <400> 218

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Page 133 s1546PCT25.5T25. txt

Ser Arg Lys Phe Lys His Glu val Ser Leu Thr phe Asp val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 . val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 ] 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 ! Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160 ; Lys Ser Ser Pro Ala Ala Asp 165 i <210> 219 ’ : <211> 167 <212> PRT <213> artificial sequence <220> . } <223> Optimized GSCHO1.3 variant of Table XII <400> 219

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gin

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr GIn Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

Page 134 s1546PCT25.5T25. txt 85 90 a5

Arg Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp Ala Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 220 <211> 167 <212> PRT <213> artificial sequence <220> <223> Optimized GSCHOl1l.3 variant of Table XII <400> 220

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp Leu Thr Gln 40 45

Lys Thr Arg Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 0 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Page 135 s1546PCT25.5T25. txt

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 221 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOl1.3 variant of Table XIX <400> 221

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr GIn Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 20 95 ’

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Arg Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp Ala Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr ’ 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 222 <211> 167 <212> PRT <213> artificial sequence

Page 136 oo ] s1546PCT25.5T25. txt <220> i <223> optimized GSCHOl.3 variant of Table XII <400> 222

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 :

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly . 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr GIn Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Arg Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gin Ala Asn Leu val Leu Lys Ile Ile Glu Glin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 223 ' <211> 167 ’ <212> PRT <213> artificial sequence <220> . <223> optimized G5CHO1.3 variant of Table XII <400> 223

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr phe Asp val Thr Gln

Page 137 s1546PCT25.5T25. txt 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gin Pro Phe Leu 85 90 95 :

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 ’ 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser GTu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Pro Ser Pro Ala Ala Asp 165 <210> 224 : . <211> 167 <212> PRT, <213> artificial sequence <220> . <223> optimized GSCHO1.3 variant of Table XII <400> 224

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val -Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Page 138 s1546PCT25.5T25. txt

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Glu Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 225 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOL1.3 variant of Table XII <400> 225

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr phe Asp val Thr Gln 40 45

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Leu Leu Thr Gln Leu Gln Pro Phe Leu 85 a0 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Arg Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys

Page 139

$1546PCT25.5T25. txt 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 226 <211l> 167 <212> PRT <213> artificial sequence <220> : <223> Optimized GSCHOl.3 variant of Table XII <400> 226

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Ala Leu Lys ITe Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys : 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp ; 165 <210> 227 <211> 167 <212> PRT <213> artificial sequence <220> <223> Optimized GSCHOl.3 variant of Table XII

Page 140

} s1546PCT25.5T25. txt <400> 227

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg GIn ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45 :

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60

Ala Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Ile Leu Thr GIn Leu GIn Pro Phe Leu 35 20 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Ala Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Val Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asn Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys : 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 228 <211> 167 <212> PRT <213> artificial sequence <220> } <223> Optimized GSCHO1l.3 variant of Table XII <400> 228

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 rhe val Asp Gly Asp Gly Ser Ile val Ala Gln Ile Lys Pro Arg Gln 20 25 30 ser Tyr Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 35 . 40 45

Page 141 ’

s1546PCT25.5T25. txt

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 229 <211> 167 <212> PRT <213> artificial sequence <220> <223> Optimized GSCHO1l.3 variant of Table XII <400> 229

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Thr val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Asn Tyr Ile Leu Ser 65 70 75 30

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln

Page 142

51546PCT25.5T25. txt 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 230 . <211> 33 : <212> DNA . <213> artificial sequence <220> <223> G19SF primer <400> 230 : gccggctttg tggactctga cggtagcatc atc 33 <210> 231 <211> 33 <212> DNA <213> artificial sequence <220> <223> G19SR primer <400> 231 gatgatgcta ccgtcagagt ccacaaagcc ggc 33 <210> 232 <211> 33 <212> DNA . <213> artificial sequence <220> . <223> FD54LF primer <400> 232 acccagcgcc gttggctgct ggacaaacta gtg | 33 <210> 233 <211> 33 <212> DNA <213> artificial sequence <220> <223> F54LR primer <400> 233 cactagttitg tccagcagcc aacggcgetg ggt 33 <210> 234

Page 143

~ s1546PCT25.5T25. txt <211> 33 : <212> DNA <213> artificial sequence <220> <223> EBOKF primer <400> 234 ttaagcaaaa tcaagccgct gcacaacttc ctg 33 <210> 235 <211> 33 <212> DNA <213> artificial sequence <220> } <223> EBOKR primer <400> 235 caggaagttg tgcagcggct tgattttgct taa 33 <210> 236 <211> 33 <212> DNA <213> artificial sequence <220> <223> F87LF primer <400> 236 aagccgctge acaacctgct gactcaactg cag 33 <210> 237 <211> 33 <212> DNA <213> artificial sequence <220> } <223> FB7LR primer <400> 237 ctgcagttga gtcagcaggt tgtgcagcgg ctt 33 <210> 238 <211> 33 . <212> DNA <213> artificial sequence <220> . <223> VI105AF primer . <400> 238 aaacaggcaa acctggctct gaaaattatc gaa 33 <210> 239 <211> 33 <212> DNA . <213> artificial sequence <220> <223> VI105AR primer <400> 239

Page 144

51546PCT25.5T25. tXt ttcgataatt ttcagagcca ggtttgectg ttt 33 <210> 240 <211> 33 <212> DNA <213> artificial sequence <220> <223> 1I132VF primer <400> 240 acctgggtgg atcaggttgc agctctgaac gat 33 <210> 241 <211> 33 <212> DNA <213> artificial sequence <220> <223> 1I132VR primer <400> 241 atcgttcaga gctgcaacct gatccaccca ggt 33 <210> 242 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOLl.3 variant Table XIX <400> 242

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly: 1 5 10 15

Phe val Asp Ser Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val cys Thr 115 120 125

Page 145

51546PCT25.5T25. txt

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 243 . <211> 167 <212> PRT <213> artificial sequence <220> C <223> optimized GSCHO1.3 variant Table XII . <400> 243

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr : 115 120 125

Trp val Asp Gln val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 244

Page 146 s1546PCT25.5T25. txt <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOl.3 variant Table XII <400> 244

Met Ala Ash Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr Phe Asp val Thr Gln : 35 40 45 .

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr GIn Leu Ser 65 70 75 80 _ Lys Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu : 85 90 95

Lys Leu Lys GIn Lys Gln Ala Ash Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 245 <211> 167 : <212> PRT : <213> artificial seqgeunce <220> <223> optimized GSCHOLl.4 variant Table XIII i <400> 245 .

Met Ala Asn Ala Lys Tyr Ash Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Page 147

§1546PCT25.5T25. txt

Ser Arg Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu ITe Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 } 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr } 115 120 125 }

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 246 <211s 167 <212> PRT <213> artificial sequence <220> CL } <223> optimized GSCHOl.4 variant Table XIII <400> 246

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 35 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Page 148 s1546PCT25.5T25. txt

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Leu Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pra Ala Ala Asp 165 <210> 247 <211> 167 <212> PRT . <213> artificial sequence <220> . <223> optimized GSCHOl.4 variant Table XIII <400> 247

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Ser Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Asp Tyr Lys Phe Lys His Gln Leu Ser Leu Thr phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 90 a5

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gin Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr

Page 149 s1546PCT25.5T25. txt 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys - 145 150 155 160

Lys Thr Ser Pro Ala Ala Asp 165 : . <210> 248 <211> 167 <212> PRT __ <213> artificial sequence <220> <223> optimized GSCHOl1.4 variant Table XIII <400> 248

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu G1n Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Gly Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp : 165 <210> 249 <211> 167 <212> PRT

Page 150

51546PCT25.5T25. txt <213> artificial sequence <220> <223> optimized GSCHOl.4 variant Table XIII <400> 249

Met Ala ser Thr Lys Tyr Ash Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 . 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser i 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu : 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 . 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 250 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1.4 variant Table XIII <400> 250

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Page 151

51546PCT25.5T25. txt

Ser Arg Lys Phe Lys His Gln Leu Ser Leu Thr phe Gln val Thr Gln 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr G1n Leu GIn Pro Phe Leu 85 20 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 251 i <211> 167 <212> PRT <213> artificial sequence : <220> . <223> optimized GSCHOLl.4 variant Table XIII <400> 251

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln ser His Lys Phe Lys His GIn Leu Ser Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr Gin Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu

Page 152

51546PCT25.5T25. txt 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr arg Lys Thr 130 135 140

Thr ser GTu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys - 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 252 <211> 167 <212> PRT <213> artificial sequence <220> : <223> optimized GSCHOl.4 variant Table XIII <400> 252

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly : 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Ile Cys Thr 115 120 125

Trp val Asp G1In val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140 page 153 s1546PCT25.5T25., txt

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Arg ser Ser Pro Ala Ala Asp 165 <210> 253 <211> 167 <212> PRT . <213> artificial sequence <220> <223> optimized GSCHOL.4 variant Table XIII <400> 253

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn

Ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Phe Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp 165 <210> 254 <211> 167 <212> PRT, . <213> artificial sequence

Page 154

S$1546PCT25.5T25. txt <220> . <223> optimized GSCHOl.4 variant Table XIII <400> 254

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu His Leu Ala Gly 1 5 10 15 :

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln -

Ala His Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn Met Thr Gln 40 45 :

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ser Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 30 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln : 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 255 <211> 167 <212> PRT _ <213> artificial sequence <220> oo <223> optimized GSCHOl.4 variant Table XIII <400> 255

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 25 30

Ser His Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln

Page 155

51546PCT25.5T25. txt 35 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Tyr Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu ITe Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 a0 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125 :

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 256 <211> 167 <212> PRT . <213> artificial sequence <220> <223> optimized GSCHOLl.4 variant Table XIII <400> 256

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr GlIn 40 45

Lys Thr Arg Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu G1n Pro Phe Leu 85 90 95

Page 156

51546PCT25.5T25. txt

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 . 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 257 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1.4 variant Table XIII : <400> 257

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 "phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His Gln Leu Ser teu Thr Phe G1n val Thr Gln i 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 i 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 . 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile val Glin 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asn Ser Leu Ser Glu Lys Lys page 157 s1546PCT25.5T25. txt 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 258 <211> 167 <212> PRT <213> artificial sequence : <220> <223> optimized GSCHO1l.4 variant Table XIII <400> 258

Met Ala Asn Thr Lys Tyr Lys Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn val Thr Gln 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 S90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln . 100 105 110

Leu Pro Pro Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr . 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 259 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1.4 variant Table XIII

Page 158 .

s1546PCT25.5725. txt <400> 259

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Leu Lys His GIn Leu Ser Leu Thr Phe GIn val Thr Gn 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu ITe Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr ) 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 260 <211> 167 <212> PRT <213> artificial sequence <220> } ) <223> optimized G5CHOL1.4 variant Table XIII <400> 260

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn 20 25 30

Gly His Lys Phe Lys His GIn Leu Ser Leu Thr Phe GIn val Thr Gln 35 40 45

Page 159

§1546PCT25.5T25. txt

Lys Thr G1n Arg Arg Trp phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ser Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Tyr Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 261 <211> 167 <212> PRT <213> artificial sequence <220> . <223> optimized GSCHO1.4 variant Table XIII <400> 261 .

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn ser His Lys Phe Lys His His Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr 61n Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 63 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro phe Leu 85 90 95

Lys Leu Lys GIn Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gin

Page 160 s1546PCT25.5T25. txt 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 262 <211> 167 <212> PRT : <213> artificial sequence <220> <223> optimized GSCHOl.4 variant Table XIII <400> 262

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His GIn Leu Ser Leu Thr Phe Gln val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Thr Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 BO

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu 85 20 95 .

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn ITe Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Page 161

$1546PCT25.5T25. txt

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 263 <211> 167 . ‘ <212> PRT <213> artificial sequence : <220> : <223> optimized GSCHO1.4 variant Table XIV <400> 263

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Ser Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe GIn val Thr GIn 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asp Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asp Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 : <210> 264 <211> 167 <212> PRT <213> artificial sequence <220> C. . <223> optimized GSCHOl.4 variant Table XIV <400> 264

Page 162 s1546PCT25.5T25. txt

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Ser Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Met Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr : 130 135 140

Thr Ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 265 <211l> 167 : <212> PRT <213> artificial sequence : <220> . <223> optimized GSCHOl.4 variant Table XIV <400> 265 met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Glin 20 25 30 ser His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln val Thr Gln 35 40 45

Lys Thr G1n Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly

Page 163

51546PCT25,5T25. txt 50. 55 60 } val Gly Tyr val Ala Asp Arg Gly Ser val Ser Asp Tyr Arg Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Ash Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu val Leu Lys Ile Ile Glu Gln - 100 } 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp : 165 <210> 266 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOl.4 variant Table XIV <400> 266

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln 20 . 25 30

Gly Tyr Lys Phe Lys His GIn Leu Ser Leu Thr Phe Arg val Thr GIn 40 45

Lys Thr GIn Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gin Leu Gln Pro Phe Leu 85 90 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Page 164

51546PCT25.ST25. txt

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val asp Gln val Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr Ser Glu Thr val arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 15 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 267 <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHOL.4 variant Table xiv <400> 267

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala GIn Ile Lys Pro Arg Gln

Gly Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Leu Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 : val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser } 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu Gln Pro Phe Leu 85 20 : 95

Lys Leu Lys GIn Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu GIn 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp GIn Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser ser Pro Ala Ala Asp

Page 165 s1546PCT25.5T25. txt 165 <210> 268 <21i> 167 <212> PRT . <213> artificial sequence <220> <223> optimized GSCHOl.4 variant Table XIV <400> 268

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15

Phe val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln ser Thr Lys Phe Lys His Arg Leu Ser Leu Thr Phe Lys val Thr Gln 40 45

Lys Thr GIn Arg Arg Trp Leu Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val His Asp Arg Gly Ser val Ser Asp Tyr Ile Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr GIn Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys GIn Ala Asn Leu val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys Ser Ser Pro Ala Ala Asp 165 <210> 269 <211> 77 <212> DNA <213> artificial sequence <220> . <223> attBl-ICreIFor primer <400> 269 ggggacaagt ttgtacaaaa aagcaggctt cgaaggagat agaaccatgg ccaataccaa 60

Page 166

$1546PCT25,5T25. txt dtataacaaa gagttcc 77 <210> 270 <211> 64 } <212> DNA . <213> artificial sequence <220> <223> attB2-ICrelIRev primer <400> 270 ggggaccact ttgtacaaga aagctgggtt tagtcggccg ccggggagga trtcttcttc 60 tcge 64 <210> 271 . <211> 167 <212> PRT <213> artificial sequence <220> <223> optimized GSCHO1.3 variant of Table xv <400> 271

Met Ala Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly 1 5 10 15 '

Phe val Asp Ala Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg GIn : 20 25 30 ser Arg Lys Phe Lys His Glu Leu Ser Leu Thr phe Asp val Thr Gln 40 45

Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu val Asp Glu Ile Gly 50 55 60 val Gly Tyr val Tyr Asp Ser Gly Ser val Ser Tyr Tyr Gln Leu Ser 65 70 75 80

Glu Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu GIn Pro Phe Leu 85 90 95

Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln 100 105 110

Leu Pro Ser Ala Lys Glu Ser Pro Ala Lys Phe Leu Glu val Cys Thr 115 120 125

Trp val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr 130 135 140

Thr ser Glu Thr val Arg Ala val Leu Asp Ser Leu Ser Glu Lys Lys 145 150 155 160

Lys ser Ser Pro Ala Ala Asp

Page 167 s1546PCT25.5T25. txt 165 <210> 272 <211> 9282 <212> DNA <213> Homo sapiens <400> 272 ggtgcgggge tgctggcgge tctgcagagt cgagagtggg agaagagcgg agcgtgtgag 60 cagtactgcg gcctcctetce ctctcctaac ctcgetcteg cggcctagct ttacccgccc 120 gcctgetcgg cgaccaggta agcecccgga cggeccggtg tcacgcaage gaggegegec 180 gccectgeta ccccgecgagg cgcgccgocc agoctctttt tteggotgee tgeccctceca 240 gtcccagccc tacgctgcgg cctcteegge ccatgcctga gatccggcat gagtgetect 300 ccecgegtget tecgecgetg gtggettgga ccegteggag ctggcgctgg tggggcgcge 360 ccttggccag gctctgggaa gggcggggtg agttgttttg atcttctttt cactacttge 420 ggccgaageg ccgcccctgg aggccgttgg gccggocctge gecctgggge tcgcagtggt 480 ttgtttgcgc tgtggatgga gtggcggtge ggtcccctgt ggagcgcaaa caaggcegctt 540 ggttggcgcg ggcgcctgge tgccttcctc gtggtggggc cttcggagca atcgtcctgg 600 ttctggegat ggttgagacg cctcgattge ggcgtgtaac ggtgagcgtt gtttgggegg 660 ccggcteceg cctcggggte ccgggggect tccaatgtga ccgaacaatg gagagcccgg 720 gcctcggege agtcagtgga gaagecggtc cgggecggagg cagcagcagc gcgcagtccc 780 tacggcctge gececcccacce tcccceggac cccccaacce cteggeggca gggtatggee 840 acctccgtga ggccctgaga ttcggacggg ggcccgaggg gcagggcgce cactttaggg 900 acatttcagt gggaaggggc tgctctcaaa gtggataatt ataactccct cgggggcgag 960 aagcggggat cctcccccag ccgcaagtcc acgaagaaag caacgaatga aaattatgaa 1020 gacaacgaga agtcagactc ctccgggtcg cgctccagct gottcggett cgtcgcctac 1080 tctgtgaact ccggggagag atctcgagtc aagattaaga ccttaaccca ccaacctgec 1140 tgttcggaca ccccccggge cggccgetgt ctgtecccctt ctccatcgee ctcteccaga 1200 aagctccggt gcttggacca gctagagtct gagaaagagg agaggcgcga acgccactcc 1260 aaaaagagaa gggqttaaaga gggcaaccct aacgatacgc ttgactttct gtggctgggg 1320 tgagtgaggg ggcagggagg acgaccccgg agttggtggg agctgcagaa actgctgaaa 1380 acttcagaat ccatttcccc cacgtaaact tggcaccgca gcagcagcag ttgatagagt 1440 ggcactaggc tgctggcatg caactcggct cacggaaaag agcaagagat ccgaaactga 1500 ggcttaggac aaagtgtgca tgatattggt ggtgtaacat gttggagagg acagccgaga 1560 aattgggttg taggtttttt ttttttgttt tccgacagag tctctatctg ttacccaggt 1620 tggagtgcag tggtgcaatc tcccggctca ctgcaacctc tgocctctggg gttcaggega 1680 ttctcctgtg tcagectcec gagtagetgg gattacaggc gtgcacaacc acgcccgget 1740 aatttttgta tttttagtag agacgggagt ttcatcgtgt tggccaggct ggtctcgaac 1800

Page 1638 -

51546PCT25.5T25. txt tttgtacctc aggtgatctg cccgcttcgg tgggttgtag cttttagcgg gagctaaagg 1860 gttctgggat ggaggtggga agtaggattt ggccggctga gtcttctaga ggcaatattg 1920 gggtgttyggt cttccccaag gggaagggtg gtgaggctga gggagaagca gttatgtttt 1980 : cagctgggca aacatggacg gttgcccgta gaaactttge cactgtactt cagaacgttg 2040 ccctagtcgt tggaggagaa caatgtgttc cctttctage caccctggtc cacggaggga 2100 ggagggagag agagaatgtt tcctcttcgg ctgttgtggc ttgagagttt ctcttctttc 2160 ggaggttttg tggtagtggc tggcagttat tagtatgcct catggetttt aaatttccag 2220 atctttttga tttagaagtg gaaatttcct aattacttga caagtcttgt agaattccat 2280 aatgttgtga ttcttgccca ctatcttaag tgagacttig catgagacct atggaaatta 2340 tggcagcatt ccccttagaa tctggcttga atcagctttt gtgtaggaaa ctgctagect 2400 tctaaaaaaa tatattgtaa ccttgtttca tcctcaaatc taaatgtgta atgagttttc 2460 ttttggtggg gaggggcggt gggtttgagt taagaccaca gctaggatga aagacaaaga 2520 gaaaaacaaa ctgtggaagc caagcctgtt ctgtggctgg attttactta tattggaaga 2580 agttctatgt tttgtaaatt tgtgtattgg ttttgatttg tttcctctga tagtttagta 2640 tttggatagt ttagtgttaa cctcagctac actgaaggaa tagaccttag tcctcacaag 2700 tataagttct agcttggaag cctgggttct gcagtagctg tggaacttaa gcctgtgagce 2760 tcagggatgc agaggcatty agttactacc aagggcctga tcttttcttt agcaggcatc 2820 : tgtgttaatt gtttcaaaag gtggtgatca gttttacagc ctattataaa ggagattttt 2880 gcctactata aaactaatcc ccctgaaaga gtgagtaaac ataacttttt gtgtgttgac 2940 ttccacaagg gaaggagttyg gcacttacac tctgactttt gattcagtcyg tccttcttga 3000 gccatttttyg caggggatca gtttggagtg ggcgttaaca atgttattct ttttttcttc 3060 tccagaacac cttccaccat gaccacctca gcaagttccec acttaaataa aggcatcaag 3120 caggtgtaca tgtccctgcc tcagggtgag aaagtccagg ccatgtatat ctggatcgat 3180 ggtactggag aaggactgcg ctgcaagacc cggaccctgg acagtgagcc caagtgtgtg 3240 gaaggtgaga cagcaatgtg gagtggagca catgctgggt gggatctgca gaggggtggg 3300 cagcagcctt tgactcagcc tctggattag gectctttct tctgtttgta aaggttttct 3360 aaggcagggc ttttcagact ttattcagtc aacattaagc tcctacactg cctcaaagca 3420 gagcgacgat ggaacccttt atttcaatgg aattgtgcac gtaggccagt gtattgaaga | 3480 aaaactaagt ctggtttatg gagagttggc atggggctta gaggttgccg acctggacat 3540 ccccacttag ctggctctaa ggcaccctca gaaaaccact gctctaacct gagaatgecca 3600 tctagtttac aaactcttag aaaactgtgt ttaatactca tatcactggc ttctagatgt 3660 gaagcaaatg ctctacaatg gtttttaaat aggactaatt tttagttgat gccacttttyg 3720 gaaattctta aactaattgc gtatccctct aggagctaca gttagattat agttgtgacc 3780 ttcatttttc agtctagaac aagccatagt cttccctctt ctggaaaggg gccagaggaa 3840

Page 169 s1546PCT25.5725. txt agtatcatat cctacctagt ttagggtagt ttacttttcc tttttgagta agtgaatgat 3900 cataatacaa agcctatatt gtgtacttgc tatgtggcag atgatagtgc acagacactg 3960 aagatacaaa gtgagagtct cgtctctgcc ttcagagaac tcagtcagct agagagacca 4020 agcagccttc aaaacagtgg gaaaggtgga taggtgataa gggagcatcc tagagtaagt 4080 catcctgcta gtcgtctgtt ccctcatcta taaaataagg acataacttg ccagaataca 4140 ctgggggcat aagaaggatg caacacatta cctaatggaa gaatcagaat ccttcactat 4200 ctcaatattt taagtgattg ataggatggt agtgataaca gaatgcttca gcttgtctcc 4260 tggaagacat ttgggaaggg agtatctgat atatttcttt taaggaattg gtacaatggt 4320 cttacttgga actcaaatag gaagggctat aagatcaggt acaggtgcca gggtatacat 4380 attaatgatg gcatttatac cttaatgaat tcctggaaaa gagatattta gagatgggaa 4440 ggtgagtgaa gggctggctg tatttgcatt gcttggaaag ctcctgtatg ttttaaatgt 4500 aattttccct ctttttgccc cagagttgcc tgagtggaat ttcgatggct ctagtacttt 4560 acagtctgag ggttccaaca gtgacatgta tctcgtgcct gctgccatgt ttcgggaccc 4620 cttccgtaag gaccctaaca agctggtgtt atgtgaagtt ttcaagtaca atcgaaggcc 4680 tgcaggtgtg ttatagcaca gctatggata cccctcctca atctgtgaat gctgtgaagg 4740 ggagggagaa gacattctga aatcagcatt gggaagacta ggcaatttca gcactatttt 4800 aagaatctga gtgattcttt tccctgaact tctgctttga ggaagagata atatggccca 4860 tctttctatg gtcttctctg ttggttgcat aaaatagcat tggatttgtc cagatctgtt 4920 tgccggtett ggagtcccca gtaacagcct tcctgcctgg aatgtaggcc aggacaaatg 4980 taaaccaatg gacaaatgtt tctcaaaaat tatagaatgg ctccaagtgc ctgagaaatg 5040 aagaataaat ctgacaacca gaagcagctg tcttgtgaat agagggttaa gtgcctggeca 5100 tttggtgctt gggaggtggc cagaatgcag ataaggtgaa agttgccctg ttctaaatcc 5160 actcccatgt gacttggttg taactgagtt tagttaaaac tgaagtcttt cagagtcttc 5220 ctacagatgt acaattaaca gcttctctca tttttctgac tcggtgatcc caagaaggcc 5280 tatactgggt cagttcatac catagtgcac acctcagttg tatagaatcc aaggactatt 5340 ctcccatcag catcggtatt cagcatctat gtctttagat ccctgatggc gtattattga 5400 ctcttttttc tagagaccaa tttgaggcac acctgtaaac ggataatgga catggtgagc 5460 aaccagcacc cctggtttgyg catggagcag gagtataccc tcatggggac agatgggcac 5520 ‘ ccctttggtt ggccttccaa cggcttccca gggccccagyg gtaagtctcc ttgggttaga 5580 ggtgaaattc ccagaagtgt ctaactgtgc aggaatgccc cttcccaggg atgggaatga 5640 ctttcagaat caagaagcaa aataatacag taaaggcgaa acagccctca catcaccaaa 5700 gtccaaaaat ggatatgaat atataaagta aggttttagg gggaacgttt ggccccactg 5760 aagctgtggt gaagaggaac tcccctattg cccctcccct gccccgcacc tgcagatgaa 5820 ggcaaggata gtgattcaag agggcaaggc ttaagggcct tctgatctct gactttggga 5880

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=. il 51546PCT25.5T25. txt : ttctetggat ttcttgactc ttagtgtttt gtcctgatge ttctgtaggt ccatattact 5940 gtggtgtggg agcagacaga gcctatggca gggacatcgt ggaggcccat taccgggcct 6000 gcttgtatgc tggagtcaag attgcgggga ctaatgccga ggtcatgcct gcccaggtaa 6060 gtatagctcc aatccatcaa tgaagaaggg taggtaggtg tacataggac ttttgctagt 6120 aagggctgct gatacaccac tcactaaccc aaaacctaag aacgggttgg agtacagqgtg 6180 agaagagaac aggtttagga gattctgagt tggagtgage agttagecttt gttttaatgg 6240 ccaagcttct cgtttctagt gggaatttca gattggacct tgtgaaggaa tcagcatggg 6300 agatcatctc tgggtggccc gtttcatctt gcatcgtgtg tgtgaagact ttggagtgat 6360 agcaaccttt gatcctaage ccattcctgg gaactggaat ggtgcaggct gccataccaa 6420 cttcagcacc aaggccatgc gggaggagaa tggtctgaag tgagtacctt ctgctgggge 6480 catctttaat ctcctgtgge agaaaacttg ggaggagact tagcaatctc tcagcaaagt 6540 ctcctttgca ggatgacttg caaatatttg ccaaagatga gtaaacttga cttctcagtc 6600 tggacgtact ttaggtgttg acacttgcct tcacattctc tcattttgtt cctatttgaa 6660 aaataccaaa taatacttct gattcacagt gataaatatt tgttataatt tatataatat 6720 atattagtca tatatcatta tataaatata tatcgatata tatatttgtg acatatgtca 6780 tggtgacagg gaaaagttga caaattcatg catttgaaaa tcttttagaa ctaaattagt 6840 aacaatacag gcatgtggat aagcttaatg cttatgaggg ggagaaagtt tcaaatgatt 6900 agtcttttca acaaacagta actttgtact gcttgtcggg cactgttctc accactgaga 6960 cacacaggta agaagatgca gccactgccc tcatgaagta tttgttctac tggtatcata 7020 ttttggtgeca cttcattctt ggctccatac ctggagacaa ggttggactg ccatcttttc 7080 tgtttactct aggtacatcg aggaggccat tgagaaacta agcaagcgge accagtacca 7140 catccgtgce tatgatccca agggaggect ggacaatgcc cgacgtctaa ctggattcca 7200 tgaaacctec aacatcaacg acttttctge tggtgtagcc aatcgtagcg ccagcatacg 7260 cattccccgg actgttggcc aggagaagaa gggttacttt gaagatcgtc gcccctctge 7320 caactgcgac ccettttegg tgacagaagc cctcatccge acgtgtcttc tcaatgaaac 7380 cggcgatgag cccttccagt acaaaaatta agtggactag acctccagct gttgagcccc 7440 tcctagttct tcatcccact ccaactcttc cccctctcce agttgteccg attgtaactc 7500 aaagggtgga atatcaaggt cgtttttttc attccatgtg cccagttaat cttgctttct 7560 ttgtttggct gggatagagg ggtcaagtta ttaatttctt cacacctacc ctcctttttt 7620 tcectatcac tgaagetttt tagtgcatta gtggggagga gggtggggag acataaccac 7680 tgcttccatt taatggggtg cacctgtcca ataggcgtag ctatccggac agagcacgtt 7740 tgcagaaggg ggtctcttct tccaggtagc tgaaagggga agacctgacg tactctggtt 7800 aggttaggac ttgccctcgt ggtggaaact tttcttaaaa agttataacc aactttteta 7860 ttaaaagtgg gaattaggag agaaggtagg ggttgggaat cagagagaat ggctttggtc 7920

Page 171 s1546PCT25.5T25. txt tcttgettgt gggactagcc tggettggga ctaaatgccc tgctctgaac acgaagetta 7980 gtataaactg atggatatcc ctaccttgaa agaagaaaag gttcttactg cttggtcctt 8040 gatttatcac acaaagcaga atagtatttt tatatttaaa tgtaaagaca aaaaactata 8100 tgtatggttt tgtggattat gtgtgttttg ctaaaggaaa aaaccatcca ggtcacgggg 8160 caccaaattt gagacaaata gtcggattag aaataaagca tctcattttg agtagagagc 8220 dagggaagtg gttcttagat ggtgatctgg gattaggccc tcaagaccct tttgggtttc 8280 tgcectgece accctctgga gaaggtggge actggattag ttaacagaca acacgttact 8340 agcagtcact tgatctccgt ggctttggtt taaaagacac acttgtccac ataggtttag 8400 agataagagt tggctggtca acttgagcat gttactgaca gagggggtat tggggttatt 8460 ttctggtagg aatagcatgt cactaaagca ggccttttga tattaaattt tttaaaaagc 8520 daaattatag aagtttagat tttaatcaaa tttgtagggt ttctaggtaa tttttacaga 8580 attgcttgtt tgcttcaact gtctcctacc tctgctcttg gaggagatgg ggacagggct 8640 ggagtcaaaa cacttgtaat tttgtatctt gatgtctttg ttaagactgc tgaagaatta 8700 trttttttct tttataataa ggaataaacc ccacctttat tccttcattt catctaccat 8760 tttctggttc ttgtgttggc tgtggcaggc cagctgtggt tttcttttge catgacaact 8820 tctaattgcc atgtacagta tgttcaaagt caaataactc ctcattgtaa acaaactgtg 8880 taactgccca aagcagcact tataaatcag cctaacataa gatctctctg atgtgtttgt 8940 ~ gattctttca aatccctatg tgccattata tttctttatt tcctaaaaca ggcaaaataa 9000 gctcaagttt atgtactctg agtttttaaa acactggagt gatgttgctg accagccgtt 9060 tcctgtacct ctctaagttg ggtatttggg acttaaggga ttaagttttt cacctagact 9120 tagttacaca caatcttggc atttcctagc ctagaggttt gtagcagggt acaagcccca 9180 ctcctccocce ttectttget cccctgagtt tggttttgge ttaccataac attgttttga 9240 ccattcctag cctaatacaa tagcctaaca taatgtaaga tt 9282

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Claims (1)

1. : 1°) An I-Crel variant, characterized in that at least one of the two I- Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 28 to 40 and 44 to 77 of I-Crel, said variant being able to cleave a DNA target sequence from the Glutamine Synthetase gene, and being obtainable by a method comprising at least the steps of:

   (a) constructing a first series of 1-Crel variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 28 to 40 of I-Crel,

   (b) constructing a second series of I-Crel variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of I-Crel, (c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-Crel site wherein at least (i) the nucleotide triplet at positions -10 to -8 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions -10 to -8 of said DNA target sequence from the Glutamine Synthetase gene and (ii) the nucleotide triplet at positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions -10 to -8 of said DNA target sequence from the Glutamine Synthetase gene,

   (d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-Crel site wherein at least (i) the nucleotide triplet at positions -5 to -3 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions -5 to -3 of said DNA target sequence from the Glutamine Synthetase gene and (ii) the nucleotide triplet at positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions -5 to -3 of said DNA target sequence from the Glutamine Synthetase gene, :

   (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-Crel site wherein at least (i) the nucleotide triplet at positions +8 to +10 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions +8 to +10 of said DNA target sequence from the Glutamine Synthetase gene and (ii) the nucleotide triplet at positions -10 to -8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at position +8 to +10 of said DNA target sequence from the Glutamine Synthetase gene, (f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-Crel site wherein at least (i) the nucleotide oo triplet at positions +3 to +5 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions +3 to +5 of said DNA target sequence from the Glutamine Synthetase gene and (ii) the nucleotide triplet at positions -5 to -3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at position +3 to +5 of said DNA target sequence from the Glutamine Synthetase gene, . : (g) combining in a single variant, the mutation(s) at positions 26 to 40 and 44 to 77 of two variants from step (¢) and step (d), to obtain a novel homodimeric I-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet at positions -10 to -8 is identical to the nucleotide triplet which is present at positions -10 to -8 of said DNA target sequence from the Glutamine Synthetase gene, (ii) the nucleotide triplet at positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions -10 to - 8 of said DNA target sequence from the Glutamine Synthetase gene, (iii) the nucleotide triplet at positions -5 to -3 is identical to the nucleotide triplet which is present at positions -5 to -3 of said DNA target sequence from the Glutamine Synthetase gene and (iv) the nucleotide triplet at positions +3 to +5 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions -5t0-3 of said DNA target sequence from the Glutamine Synthetase gene, and/or (h) combining in a single vanant, the mutation(s) at positions 26 to © 40 and 44 to 77 of two variants from step (e) and step (f), to obtain a novel homodimeric I-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet at positions +3 to +5 is identical to the nucleotide triplet which is present at positions +3 to +5 of said DNA target sequence from the Glutamine Synthetase gene, (ii) the nucleotide triplet at positions -5 to -3 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions +3 to +5 of said DNA : target sequence from the Glutamine Synthetase gene, (iii) the nucleotide triplet at positions +8 to +10 of the I-Crel site has been replaced with the nucleotide triplet which is present at positions +8 to +10 of said DNA target sequence from the Glutamine Synthetase gene and (iv) the nucleotide triplet at positions -10 to -8 is identical to the reverse complementary sequence of the nucleotide triplet at positions +8 to +10 of said DNA target sequence from the Glutamine Synthetase gene, (i) combining the variants obtained in steps (g) and/or (h) to form heterodimers, and

   (j) selecting and/or screening the heterodimers from step (i) which are able to cleave said DNA target sequence from the Glutamine Synthetase gene. 2°) The variant of claim 1, wherein said substitution(s) in the subdomain situated from positions 44 to 77 of I-Crel are at positions 44, 68, 70, 75 and/or 77.

   3°) The variant of claim 1, wherein said substitution(s) in the subdomain situated from positions 28 to 40 of I-Crel are at positions 28, 30, 32, 33, 38 and/or 40.

   4°) The variant of anyone of claims 1 to 3, wherein said substitutions are replacement of the initial amino acids with amino acids selected in the group consisting of A, D,E,G, H, Kk, N, P,Q, R,S, T,Y,C, V,L,M, F, Tand W.

   5°) The variant of anyone of claims 1 to 4, which is an heterodimer, resulting from the association of a first and a second monomer having different muta- tions at positions 28 to 40 and/or 44 to 77 of I-Crel, said heterodimer being able to cleave a non-palindromic DNA target sequence from the Glutamine Synthetase gene.

   6°) The variant of claim 5, wherein said DNA target is from the human GS gene and is selected from the group consisting of the sequences SEQ ID NO: 51028.

   7°) The variant of claim 5, wherein said DNA target is from the mouse GS gene and is selected from the group consisting of the sequences SEQ ID NO: 19 and 29 to 48.

   80° 8°) The variant of claim 5, wherein said DNA target is from the Chinese Hamster GS gene and is selected from the group consisting of the sequences SEQ ID NO: 19, 29, 30, 34, 46, 47 and 49 to 60. 9°) The variant of anyone of claims 1 to 8, which comprises at least one substitution at positions 137 to 143 of I-Crel that modifies the specificity of the variant towards the nucleotide at positions £ 1 to 2, £ 6 to 7 and/or £ 11 to 12 of the I- Crel site. 10°) The variant of anyone of claims 1 to 9, which comprises at least one substitution on the entire I-Crel sequence that improves the binding and/or the cleavage properties of the variant towards said DNA target sequence from the Glutamine Synthetase gene. 11°) The variant of claim 10, which comprises at least one substitu- tion selected from the group consisting of: N2S, T3A, NeK, K7E, Y12H, G19S, ' GI19A, 124V, F351, L39V, F43L, V45L, V45M, Q47K, Q50R, F54L, K57E, V59A, D60Y, V64A, Y66H, ESOK, F87L, F87I, Q92R, K96R, V105A, K107R, E110V, S114F, S114P, E117V, S118T, P119L, D120A, DI120E, V1251, V129A, 1132V, D137N, D137Y, K139R, D153N, §154G, K160R, S161P and S161T. 12°) The variant of claim 11, which comprises at least one substitu- tion selected from the group consisting of: G19S, F54L, E80K, F87L, V105A and I132V. 13°) The variant of anyone of claims 6 and 10 to 12, wherein the first and the second monomer, respectively, have amino acids at positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75, 77, and eventually at position 80, which are selected from the group consisting of : KTSGQS/VERNR+K80 and KRTNQQ/DRSRT, KNGCAS/IRSNR and KNSRDR/YASRI, KRTCQT/KYSEV and KRTNQQ/LRNNI+K80, = KNSCAS/NTSRY and KNSTAS/MERNR, KNSRNQ/RYSEV and ENSRRK/NRSRY, KNTCQS/LRANV and KYTCQS/DYSSR, KNHHQS/NQSSV and KNSEQE/AYSYK, KRCCQE/KTINI and KNRDQS/ARSRL, KRSYQS/QESRR and KSSHKS/NY SRV, KNSTQS/ARSER and KNSPQQ/KYSEV, KDSRTS/RRSND and KNCCHS/RRSND, KNHHQS/YYSST and KTSGQS/QRSYR, KTSGQS/KRSRR and KNSRAQ/NRSRY, KNSYQS/KYSNQ and ENSRRK/KYSQN, KSSHKS/IRSNR and KNDYCS/KESDR, KNSTQT/QNSQR and KNSRAQ/AQNNI, KNSRDR/IRCNR and KRANQE/ARSER, KNSPKS/NKHNI and RNSAYQ/DYSSR, KNSCAS/IRANR and KNTCQS/QRSHY, KNTY WS/ARSRL and KNSRNQ/YSSSD, KDSRSS/YRSDV and KNTYWS/DYSSR, KTSGQS/KRSDK and NNSSRK/AYSRI, KNSRNQ/SRSYT and KDSRQS/KESDR, KNSCAS/ARSER ~ and KRSRQS/QYRNI. 14°) The variant of anyone of claims 7 and 10 to 12, wherein the first and the second monomer, respectively, have amino acids at positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75, 77, and eventually at position 24 or 80, which are selected from the group consisting of : KNCCHS/KYSNI and KRSYQS/TYSRT, KRSRES/DYSYQ and KRGYQS/RHRDI, KRGYQS/KHRDI, KRGYQS/KNRD], KRCYQS/RHRDI, KRGYQS/NYSRY, KRGYQS/RTRDI, KRGYQS/TYSRV, KRGYQS/KARDI, KRGYQS/QYSRY, KKSAQS/NYSRY, KRDYQS/QRSRT+KS80, or KRDYQS/TRSRI+K80, KHHCQT/RYSEV and KRTNQQ/IRCNR, KNSRAQ/RYSER and KNSHAS/VERNR+K80, KRGRQA/RYSER and KNRDQS/TYSRT, KNSYQS/LRNNI+K80 and KNSYQS/NYSYN+V24, KDSRQS/YRSDV and KHHCAS/DRSRQ, KNSTQS/DYSSR and’ KNSRAQ/RNSQI, KNHHQS/QHSNR and KNSGQQ/QYSRV, KDSRTS/AYSYK and KRTYQS/RSSNT, KNSCQQ/QRSNR and KNDYYS/TYSRV, KRYSQS/RYSEQ and KNSYRK/KSSNI, KNSCAS/NYSRV and KNTYQS/QHSNR, KNSSRD/QRSNI and KNSYQS/KESDR, KNTCQS/ECSNI and KNNGQS/KYSNI, KSSHKS/IRSNR and KNDYCS/KESDR, KTSHRS/NRSRY and KNTYWS/DYSSR, KNSYHS/AYSRV and KDSRGS/QNSRV, KDSRQS/KESDR and NNSYRK/ARRNI, KNSRNQ/ARSRL and KWSCQS/KASDK, KGSYKS/TRSER and KNSYGQ/KYSNQ. 15°) The variant of anyone of claims 8 and 10 to 12, wherein the first and the second monomer, respectively, have amino acids at positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75, 77, and eventually at position 24 or 80, which are selected from the group consisting of: KNCCHS/KYSNI and KRSYQS/TYRST, KRSRES/DYSYQ and KRGYQS/RHRDI, KRGYQS/KHRDI, KRGYQS/KNRDI, KRCYQS/RHRDI, KRGYQS/NYSRY, KRGYQS/RTRDI, KRGYQS/TYSRV, KRGYQS/KARDI, KRGYQS/QYSRY, KKSAQS/NYSRY, KRDYQS/QRSRT+KS0,

   or KRDYQS/TRSRI+K 80, KNHCQA/RYSER and KRTNQ/IRSNR, KNSRDR/KYSEV and KNSGQG/TYSYR, KNSNQR/KYSEV and KRDYQS/NYSYQ, KRSYQS/KESDR and KNHHQS/RYSEY, KNSYQS/LRNNI+K80 and KNSYQS/NYSYN+V24, KRSYQS/KSSNV and KSTSRS/AYSDH, KSSCQA/NKHNI and KNGHQS/QESRR, KRSYQS/QHSNR : and KTSGQS/QYSRYV, KNRDQS/ECSNI and KNSNYR/QRDNR, KSSHKS/IRSNR and KNDCQS/KESDR, KNSHQT/NRSRY and KRSYES/DYSSR, KNSCQH/VERNR+K80 and SNSYRK/NRSRY, KDSRTS/YRSDV and KKSSQS/DYSSR, KDSRQS/KESDR and NNSYRK/ARRNI, KNSRNQ/ARSRL and KWSCQS/KASDK, KGSYKS/ARSER and KNSRQR/ARGNI. 16°) The variant of anyone of claims 13 to 15, wherein the first ‘monomer and the second monomer, respectively, are selected from the following pairs of sequences: SEQ ID NO: 61 to 84 (first monomer) and SEQ ID NO: 85 to 108, (second monomer); SEQ ID NO: 109, 110, 63, 111 to 128 (first monomer) and SEQ ID NO: 129 to 134, 89, 135 to 151 (second monomer); SEQ ID NO: 109, 110, 63, 152 to 154, 113, 155 to 158, 123, 159 to 162, 127, 163 (first monomer) and SEQ ID NO: 164, 130 to 133, 198-200, 203 and 206 to 208, 134, 89,165, 166, 136, 167 to 170, 146, 147, 171 to 175 (second monomer). 17°) The variant of anyone of claims 7, 8, 10 to 12, 14 and 15, which cleaves the DNA target sequence SEQ ID NO: 30 from the mouse and Criteculus sp.

   Glutamine Synthetase gene and comprises a first and a second monomer having amino acids at positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75 and 77 and at additional positions, which are selected from the group consisting of: © KRSRES/DYSYQ+H66+132V, 'KRSRES/DYSYQ+A19+120A or KRSRES/DYSYQ+S19+E57+T1184+V132 (first monomer) and KRGQS/RHRDI, KRGYQS/QARDR+I19L or KRSRQS/QARDR (second monomer); KRSRES/DYSYQ+H66+132V (first monomer) and KRGYQS/KHRDI+S19+M45 (second monomer). 18°) The variant of anyone of claims 7, 8, 10 to 12 14, 15 and 17, which cleaves the DNA target sequence SEQ ID NO: 30 from the mouse and Criteculus sp.

   Glutamine Synthetase gene and comprises a first monomer having any of the sequence SEQ ID NO: 211 to 229, 242 to 244 and 271 and a second monomer having any of the sequence SEQ ID NO: 245 to 268. 19°) The variant of anyone of claims 1 to 15 and 17, which has at - least 95 % sequence identity with one of the sequences as defined in claim 16 or claim : 5 18. 20°) The variant of anyone of claims 1 to 19, which comprises a nuclear localization signal and/or a tag. - 219) The variant of anyone of claims 5 to 20, which is an obligate heterodimer, wherein the first monomer further comprises the D137R mutation and the second monomer further comprises thé R51D mutation. 22°) The variant of anyone of claims 5 to 20, which is an obligate heterodimer, wherein the first monomer further comprises the ESR or E8K. and E61R mutations and the second monomer further comprises the K7E and K96E mutations. 23°) A single-chain meganuclease comprising two monomers or core domains of one variant of anyone of claims 1 to 22, or a combination of both. 24°) The single-chain meganuclease of claim 23, which comprises the first and the second monomer as defined in anyone of claims 13 to 18 connected by a peptidic linker. 25°) A polynucleotide fragment encoding the variant of anyone of claims I to 22 or the single-chain meganuclease of claim 23 or claim 24. 26°) An expression vector comprising at least one polynucleotide fragment of claim 25. 27°) The expression vector of claim 26, which comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant of anyone of claims 5 to 22. 28°) The vector of claim 26 or claim 27, which includes a targeting construct comprising a sequence to be introduced in the Glutamine Synthetase gene and a sequence homologous to the sequence of the Glutamine Synthetase gene flanking the genomic DNA cleavage site of the [-Crel variant as defined in anyone of claims 1, and 5 to 8. 29°) The vector of claim 28, wherein said sequence to be introduced is a sequence which inactivates the Glutamine Synthetase gene, flanked by sequences homologous to the sequences of the Glutamine Synthetase gene flanking the genomic DNA cleavage site of the I-Crel variant. 30°) The vector of claim 28, wherein said sequence to be introduced is a sequence which repairs a mutation in the human Glutamine Synthetase gene. oo 5 31°) The vector of claim 30, wherein said sequence encodes a portion of wild-type human Glutamine Synthetase. 32°) The vector of claim 28, wherein said sequence homologous to the sequence of the Glutamine Synthetase gene flanking the genomic DNA cleavage site of the I-Crel variant comprises the sequence encoding a portion of wild-type Glutamine Synthetase as defined in claim 31. 33°) The vector of claim 30, wherein the sequence which repairs said mutation comprises the human Glutamine Synthetase open reading frame and a polyadenylation site to stop transcription in 3’, flanked by sequences homologous to the sequences of the human Glutamine Synthetase gene flanking the genomic DNA cleavage site of the I-Crel variant. 34°) A composition comprising at least one variant of anyone of claims | to 22, one single-chain meganuclease of claim 23 or claim 24, and/or one expression vector of anyone of claims 26 to 33. 35°) The composition of claim 34, which comprises a targeting DNA construct as defined in anyone of claims 28 to 33. : 36°) The composition of claim 35, wherein said targeting DNA construct is included in a recombinant vector . 37°) A host cell which is modified by at least one polynucleotide fragment as defined in claim 25 or claim 27 or one vector of anyone of claims 26 to

   33. 38°) A non-human transgenic animal comprising one or two polynucleotide fragments as defined in claim 25 or claim 27. 39°) A transgenic plant comprising one or two polynucleotide fragments as defined in claim 25 or claim 27. 40°) Use of at least one variant of anyone of claims 1 to 22, one single-chain meganuclease of claim 23 or claim 24, and/or one expression vector according to anyone of claims 26 to 33 for genome engineering, for non-therapeutic purposes. } 41°) The use of claim 40 for making Glutamine Synthetase knock-out animal/cell line.

   42°) The use of claim 41, wherein said animal/cell line is a transgenic : animal/cell line wherein the transgene is inserted at the Glutamine Synthetase gene locus.

   43°) The use of claim 41, wherein said animal/cell line is a transgenic animal/cell line wherein the transgene is inserted at any genomic locus.

   44°) Use of at least one variant of anyone of claims 1 to 22, one single-chain meganuclease of claim 23 or claim 24, and/or one expression vector according to anyone of claims 26 to 33, for the preparation of a medicament for preventing, improving or curing a pathological condition caused by a mutation in the Glutamine Synthetase gene. :

   45°) The use of anyone of claims 40 to 44, wherein said variant, single-chain meganuclease, or vector is associated with a targeting DNA construct as defined in anyone of claims 28 to 33.