Cellectis

Cellectis S.a.

Frederic Paques

Philippe Duchateau

Jean-Charles Epinat

Emmanuel Lacroix

Sylvain Arnould

Patrick Chames

Phillippe Duchateau

Andre Choulika

Julianne Smith

Agnes Gouble

Jean-Pierre Cabaniols

David Sourdive

Christophe Perez-Michaut

Sylvia Bruneau

Method of preparing I-CreI meganuclease variants having a modified cleavage specificity, variants obtainable by said method and their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes. Nucleic acids encoding said variants, expression cassettes comprising said nucleic acids, vectors comprising said expression cassettes, cells or organisms, plants or animals except humans, transformed by said vectors.

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Use of meganucleases for cleaving DNA in a non-human or an isolated human cell and, in some instances, inducing homologous recombination in said cells and to its application for genome engineering and gene therapy.

Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

A monomer of an I-CreI meganuclease variant wherein said monomer comprises mutations in the amino acid sequence of SEQ ID NO: 34, wherein said mutations include, (i) at least one and up to five amino acid substitutions from residue Q44 to residue R70, said substitutions selected from the group consisting of substitutions at positions Q44, T46, Y66, R68 and R7; and (ii) at least one and up to six amino acid substitutions from residue Q26 to residue Q38 said substitutions selected from the group consisting of substitutions at positions Q26, K28, N30, S32, Y33 and Q38, and wherein said monomer when in dimeric form binds and cleaves a DNA target sequence. Said dimeric forms include homodimeric, heterodimeric and single-chain I-CreI meganuclease variants.

The present invention relates to a polynucleotide encoding a monomer of an I-CreI variant said monomer comprising mutations in the amino acid sequence of SEQ ID NO: 34, wherein said mutations include: (i) at least one and up to eleven amino acid substitutions from residue S22 to Q44 said substitutions selected from the group consisting of substitutions at positions I24, Q26, K28, N30, S32, Y33, Q38, S40 and T42; and (ii) at least one and up to six amino acid substitutions from residue Y66 to I77 said substitutions selected from the group consisting of substitutions at positions Y66, R68, R70, V73 and I77; and wherein said monomer when in dimeric form binds and cleaves DNA.

Method of preparing 1-CreI meganuclease variants having a modified cleavage specificity, variants obtainable by said method and their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes. Nucleic acids encoding said variants, expression cassettes comprising said nucleic acids, vectors comprising said expression cassettes, cells or organisms, plants or animals except humans, transformed by said vectors.

A single chain homing endonuclease, comprising a first variant of I-CreI having the amino acid sequence of accession number pdb 1g9y (SEQ ID NO: 23 is residues 1-153 of pdb Ig9y) and a second variant of I-CreI variant having the amino acid sequence of accession number pdb 1g9y (SEQ ID NO: 23 is residues 1-153 of pdb Ig9y) in a single polypeptide.

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Bourg la Reine, France

Frederic Paques