RATIONALLY-DESIGNED MEGANUCLEASES WITH ALTERED SEQUENCE SPECIFICITY AND DNA-BINDING AFFINITY
First Claim
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1. A method of cleaving a target recognition sequence in double-stranded DNA, 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, including a substitution corresponding to position −
8 of the I-CreI recognition site, the method comprising;
(a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease,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; and
wherein;
(i) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33L, Y33V, Y33I, Y33F and Y33C;
(ii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to an C on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33E, Y33D and 532R;
(iii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33F, Y33H and Y33R; and
(b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence.
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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.
3 Citations
15 Claims
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1. A method of cleaving a target recognition sequence in double-stranded DNA, 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, including a substitution corresponding to position −
8 of the I-CreI recognition site, the method comprising;(a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease, 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; andwherein; (i) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33L, Y33V, Y33I, Y33F and Y33C;(ii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to an C on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33E, Y33D and 532R;(iii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33F, Y33H and Y33R; and(b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence. - View Dependent Claims (14)
- 4, including a substitution corresponding to position −
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2. A method of cleaving a target recognition sequence in double-stranded DNA, wherein the target sequence differs by at least one nucleotide modification in the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:
- 4, including a substitution corresponding to position −
8 of the I-CreI recognition site, the method comprising;(a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease, 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; (i) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33L, Y33V, Y33I, Y33F and Y33C;(ii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to an C on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33E, Y33D and 532R;(iii) if the nucleotide corresponding to position −
8 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
1 selected from the group consisting of Y33F, Y33H and Y33R; andwherein; the recombinant meganuclease comprises at least a second specificity-altering amino acid modification corresponding to a substitution in 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, 540C, S40E, S40I, S40Q, S40R, 540V, 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; and (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15)
- 4, including a substitution corresponding to position −
Specification