I-CreI meganuclease variants with modified specificity, method of preparation and uses thereof
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First Claim
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1. An isolated monomer of an I-CreI meganuclease variant comprising at least one mutation in the amino acid sequence of SEQ ID NO:
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,wherein said monomer when in a dimeric form is able to cleave DNA.
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Abstract
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.
27 Citations
54 Claims
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1. An isolated monomer of an I-CreI meganuclease variant comprising at least one mutation in the amino acid sequence of SEQ ID NO:
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
wherein said monomer when in a dimeric form is able to cleave DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
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19. A purified monomer of an I-CreI meganuclease variant comprising at least one mutation in the amino acid sequence of SEQ ID NO:
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
wherein said monomer when in a dimeric form is able to cleave DNA. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
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37. A recombinant monomer of an I-CreI meganuclease variant comprising at least one mutation in the amino acid sequence of SEQ ID NO:
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
wherein said monomer when in a dimeric form is able to cleave DNA. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
- 70, wherein said at least one mutation comprises a substitution at one or more of the amino acids residues at positions 44, 68 and 70 and said monomer further comprises at least one additional mutation of an amino acid residue directly contacting a DNA target sequence wherein said amino acid residue directly contacting a DNA target sequence is selected from the group consisting of positions 26, 28, 30, 32, 33 and 38,
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Litigation Data
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Current AssigneeCellectis SA
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Original AssigneeCellectis SA
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InventorsPaques, Frederic, Duchateau, Philippe
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Primary Examiner(s)Fronda; Christian L
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Application NumberUS11/908,798Publication NumberTime in Patent Office1,812 DaysField of SearchNoneUS Class Current435/199CPC Class CodesA61K 38/00 Medicinal preparations cont...A61K 48/00 Medicinal preparations cont...A61P 31/00 Antiinfectives, i.e. antibi...A61P 31/12 AntiviralsA61P 43/00 Drugs for specific purposes...C12N 9/22 Ribonucleases RNAses, DNAse...
