METHODS OF PRODUCING GENETICALLY-MODIFIED EUKARYOTIC CELLS WITH RATIONALLY-DESIGNED MEGANUCLEASES
First Claim
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1. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
- introducing into the cell;
(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;
wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1 by at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;
1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139;
wherein the first specificity-altering amino acid modification is selected from the group consisting of;
I24C, I24K, I24R, Q26A, Q26E, Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33H, Y33I, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D, T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68c, R68E, R68F, R68H, R68K, R68L, R68M, R68Q, R68Y, R70A, R70C, R70D, R70E, R70G, R70H, R70K, R70L, R70Q, R70S, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S79I, S79V, K139H and K139Y;
wherein the DNA-binding affinity of the recombinant meganuclease has been altered compared to the DNA-binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
1, and wherein the polypeptide further comprises at least one modification affecting DNA-binding affinity corresponding to a substitution in the polypeptide of SEQ ID NO;
1 selected from the group consisting of;
P29D, P29E, P29H, P29K, P29N, P29Q, P29R, P29S, P29T, K34D, K34E, K34H, K34N, K34Q, K34S, K34T, K48D, K48E, K48H, K48N, K48Q, K48S, K48T, R51D, R51E, R51H, R51N, R51Q, R51S, R51T, V64D, V64E, V64H, V64K, V64N, V64Q, V64R, V64S, V64T, E80H, E80K, E80N, E80Q, E80R, E80S, E80T, I81D, I81E, I81H, I81K, I81N, I81Q, I81R, I81S, I81T, K82D, K82E, K82H, K82N, K82Q, K82S, K82T, L112D, L112E, L112H, L112K, L112N, L112Q, L112R, L112S, L112T, K116D, K116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R; and
wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome.
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Abstract
Rationally-designed LAGLIDADG meganucleases and methods of making such meganucleases are provided. In addition, methods are provided for using the meganucleases to generate recombinant cells and organisms having a desired DNA sequence inserted into a limited number of loci within the genome, as well as methods of gene therapy, for treatment of pathogenic infections, and for in vitro applications in diagnostics and research.
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Citations
27 Claims
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1. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
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introducing into the cell;
(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1 by at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;
1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139;wherein the first specificity-altering amino acid modification is selected from the group consisting of; I24C, I24K, I24R, Q26A, Q26E, Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33H, Y33I, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D, T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68c, R68E, R68F, R68H, R68K, R68L, R68M, R68Q, R68Y, R70A, R70C, R70D, R70E, R70G, R70H, R70K, R70L, R70Q, R70S, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S79I, S79V, K139H and K139Y; wherein the DNA-binding affinity of the recombinant meganuclease has been altered compared to the DNA-binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
1, and wherein the polypeptide further comprises at least one modification affecting DNA-binding affinity corresponding to a substitution in the polypeptide of SEQ ID NO;
1 selected from the group consisting of;P29D, P29E, P29H, P29K, P29N, P29Q, P29R, P29S, P29T, K34D, K34E, K34H, K34N, K34Q, K34S, K34T, K48D, K48E, K48H, K48N, K48Q, K48S, K48T, R51D, R51E, R51H, R51N, R51Q, R51S, R51T, V64D, V64E, V64H, V64K, V64N, V64Q, V64R, V64S, V64T, E80H, E80K, E80N, E80Q, E80R, E80S, E80T, I81D, I81E, I81H, I81K, I81N, I81Q, I81R, I81S, I81T, K82D, K82E, K82H, K82N, K82Q, K82S, K82T, L112D, L112E, L112H, L112K, L112N, L112Q, L112R, L112S, L112T, K116D, K116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R; and wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
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introducing into the cell;
(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1 by at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;
1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139;wherein the first specificity-altering amino acid modification is selected from the group consisting of; I24C, I24K, I24R, Q26A, Q26E, Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33H, Y33I, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D, T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68c, R68E, R68F, R68H, R68K, R68L, R68M, R68Q, R68Y, R70A, R70C, R70D, R70E, R70G, R70H, R70K, R70L, R70Q, R70S, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S79I, S79V, K139H and K139Y; wherein the polypeptide further comprises a second amino acid modification affecting heterodimer formation selected from the group consisting of; K7D, K7E, E8K, E8R, K57D, K57E, E61K, E61R, K96D and K96E; and wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome. - View Dependent Claims (8, 9, 10, 11, 12)
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13. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
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introducing into the cell;
(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1 by at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;
1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139;wherein the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO;
4; - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
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introducing into the cell;
(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
1 by at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;
1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139;wherein the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO;
4;
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Specification