Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
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First Claim
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1. A recombinant meganuclease comprising a polypeptide which has at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO:
- 1, wherein the DNA-binding affinity of said 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 said polypeptide 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.
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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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20 Claims
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1. A recombinant meganuclease comprising a polypeptide which has at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO:
- 1, wherein the DNA-binding affinity of said 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 said polypeptide 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. - View Dependent Claims (4)
- 1, wherein the DNA-binding affinity of said recombinant meganuclease has been altered compared to the DNA binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
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2. A recombinant meganuclease comprising a polypeptide which has at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO:
- 1, wherein the DNA-binding affinity of said 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 said polypeptide 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 said polypeptide further comprises at least one cleavage specificity-altering modification corresponding to a substitution in the polypeptide of SEQ ID NO;
1 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, Q38I, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q44I, 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. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
- 1, wherein the DNA-binding affinity of said recombinant meganuclease has been altered compared to the DNA binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
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3. A recombinant meganuclease comprising a polypeptide which has at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO:
- 1, wherein the DNA-binding affinity of said 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 said polypeptide 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;E80H, E80K, E80N, E80Q, E80R, E80S, E80T, I81H, I81K, I81N, I81Q, I81R, I81S and I81T; and wherein said polypeptide further comprises at least one cleavage specificity-altering modification corresponding to a substitution in the polypeptide of SEQ ID NO;
1 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, Q38I, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q44I, 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.
- 1, wherein the DNA-binding affinity of said recombinant meganuclease has been altered compared to the DNA binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
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Litigation Data
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Current AssigneeDuke University
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Original AssigneeDuke University
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InventorsSmith, James Jefferson, Jantz, Derek, Hellinga, Homme W.
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Primary Examiner(s)RAMIREZ, DELIA M
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Application NumberUS13/245,596Publication NumberTime in Patent Office148 DaysField of Search435/6.1, 435/69.1, 435/400, 435/196, 435/320.1, 435/325, 530/350, 536/23.2US Class Current435/196CPC Class CodesA61K 38/465 acting on ester bonds (3.1)...A61K 48/00 Medicinal preparations cont...A61P 3/00 Drugs for disorders of the ...A61P 31/00 Antiinfectives, i.e. antibi...A61P 31/12 AntiviralsA61P 43/00 Drugs for specific purposes...C12N 15/8213 Targeted insertion of genes...C12N 15/902 using homologous recombinationC12N 15/905 in yeastC12N 15/907 in mammalian cellsC12N 9/22 Ribonucleases RNAses, DNAse...
