I-CREI MEGANUCLEASE VARIANTS WITH MODIFIED SPECIFICITY, METHOD OF PREPARATION AND USES THEREOF
First Claim
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1. A method of preparing at least one I-CreI meganuclease variant having a modified cleavage specificity, said method comprising:
- (a) replacing amino acids Q44, R68 and/or R70, in reference with I-CreI pdb accession code 1g9y, with an amino acid selected from the group consisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y to obtain one or more I-CreI meganuclease variants; and
(b) selecting said one or more I-CreI meganuclease variants obtained in (a) having at least one of the following R3 triplet cleaving profile in reference to positions −
5 to −
3 in a double-strand DNA target, said positions −
5 to −
3 corresponding to R3 of the following formula I;
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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.
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Citations
58 Claims
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1. A method of preparing at least one I-CreI meganuclease variant having a modified cleavage specificity, said method comprising:
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(a) replacing amino acids Q44, R68 and/or R70, in reference with I-CreI pdb accession code 1g9y, with an amino acid selected from the group consisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y to obtain one or more I-CreI meganuclease variants; and (b) selecting said one or more I-CreI meganuclease variants obtained in (a) having at least one of the following R3 triplet cleaving profile in reference to positions −
5 to −
3 in a double-strand DNA target, said positions −
5 to −
3 corresponding to R3 of the following formula I; - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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32-35. -35. (canceled)
Specification
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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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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current424/94.600
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CPC 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...
