Methods of cleaving DNA with rationally-designed meganucleases

US 8,133,697 B2
Filed: 09/27/2011
Issued: 03/13/2012
Est. Priority Date: 10/18/2005
Status: Active Grant

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1. A method of cleaving a target recognition site in double-stranded DNA in a non-human cell or an isolated human cell, wherein said target recognition site has a sequence that differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:

4;

Position −

9-8-7-6-5-4-3-2-1 5′

-C A A A C T G T C G T G A G A C A G T T T G-3′

(SEQ ID NO;

4)wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −

5 of the I-CreI recognition site of SEQ ID NO;

4, the method comprising;

(a) introducing into a cell (i) a recombinant meganuclease, or (ii) a nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in said 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; and

wherein;

(i) if the nucleotide corresponding to position −

5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;

4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of A42Q, A42R, Y66M and Y66K;

(ii) if the nucleotide corresponding to position −

5 of the I-CreI meganuclease recognition sequence has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of A42Q, Y66M, and Y66K;

(iii) if the nucleotide corresponding to position −

5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;

4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of Y66M and Y66K; and

(b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.

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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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24 Claims

1. A method of cleaving a target recognition site in double-stranded DNA in a non-human cell or an isolated human cell, wherein said target recognition site has a sequence that differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:
- 4;
  
  Position −
  
  9-8-7-6-5-4-3-2-1 5′
  
  -C A A A C T G T C G T G A G A C A G T T T G-3′
  
  (SEQ ID NO;
  
  4)wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −
  
  5 of the I-CreI recognition site of SEQ ID NO;
  
  4, the method comprising;
  
  (a) introducing into a cell (i) a recombinant meganuclease, or (ii) a nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in said 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; and
  
  wherein;
  
  (i) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of A42Q, A42R, Y66M and Y66K;
  
  (ii) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of A42Q, Y66M, and Y66K;
  
  (iii) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of Y66M and Y66K; and
  
  (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.

2. A method of cleaving a target recognition site in double-stranded DNA in a non-human cell or an isolated human cell, wherein said target recognition site has a sequence that differs by at least one nucleotide modification in the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:
- 4;
  
  Position −
  
  9-8-7-6-5-4-3-2-1 5′
  
  -C A A A C T G T C G T G A G A C A G T T T G-3′
  
  (SEQ ID NO;
  
  4)wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −
  
  5 of the I-CreI recognition site of SEQ ID NO;
  
  4, the method comprising;
  
  (a) introducing into a cell (i) a recombinant meganuclease, or (ii) a nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in said 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;
  
  (i) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of A42Q, A42R, K28C, Y66M and Y66K;
  
  (ii) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4 has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of K28C, A42Q, Y66M, and Y66K;
  
  (iii) if the nucleotide corresponding to position −
  
  5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of Y66M, and Y66K; and
  
  wherein;
  
  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, 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, R705, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S791, S79V, K139H and K139Y; and
  
  (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.
- View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
- - 3. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a G and the first specificity-altering amino acid modification corresponds to a substitution of A42Q in SEQ ID NO;
      
      1.
  - 4. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a G and the first specificity-altering amino acid modification corresponds to a substitution of A42R in SEQ ID NO;
      
      1.
  - 5. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site has been altered to a G and the first specificity-altering amino acid modification corresponds to a substitution of K28C in SEQ ID NO;
      
      1.
  - 6. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a G and the first specificity-altering amino acid modification corresponds to a substitution of Y66M in SEQ ID NO;
      
      1.
  - 7. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a G and the first specificity-altering amino acid modification corresponds to a substitution of Y66K in SEQ ID NO;
      
      1.
  - 8. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the 1-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to an A and the first specificity-altering amino acid modification corresponds to a substitution of K28C in SEQ ID NO;
      
      1.
  - 9. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to an A and the first specificity-altering amino acid modification corresponds to a substitution of A42Q in SEQ ID NO;
      
      1.
  - 10. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to an A and the first specificity-altering amino acid modification corresponds to a substitution of Y66M in SEQ ID NO;
      
      1.
  - 11. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to an A and the first specificity-altering amino acid modification corresponds to a substitution of Y66K in SEQ ID NO;
      
      1.
  - 12. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a T and the first specificity-altering amino acid modification corresponds to a substitution of Y66M in SEQ ID NO;
      
      1.
  - 13. The method of claim 2 wherein the nucleotide corresponding to position −
    - 5 of the I-CreI meganuclease recognition site of SEQ ID NO;
      
      4 has been altered to a T and the first specificity-altering amino acid modification corresponds to a substitution of Y66K in SEQ ID NO;
      
      1.
  - 14. The method of claim 2 wherein said cell is a plant cell.
  - 15. The method of claim 14 wherein said nucleic acid is introduced into said cell by an Agrobacterium vector.
  - 16. The method of claim 14 wherein said cell is a protoplast and said nucleic acid is introduced into said cell by PEG-mediated transformation.
  - 17. The method of claim 14 wherein said nucleic acid is introduced into said cell by a method selected from the group consisting of ballistic injection and microprojectile bombardment.
  - 18. The method of claim 14 wherein said nucleic acid is introduced into said cell by electroporation.
  - 19. The method of claim 2 wherein said cell is a mammalian cell.
  - 20. The method of claim 19 wherein the nucleic acid is introduced into said cell by an adenovirus or adeno-associated virus vector.
  - 21. The method of claim 19 wherein the nucleic acid is introduced into said cell by microinjection.
  - 22. The method of claim 19 wherein the nucleic acid is introduced into said cell by a method selected from the group consisting of ballistic injection and microprojectile bombardment.
  - 23. The method of claim 19 wherein the nucleic acid is introduced into said cell by electroporation.
  - 24. The method of claim 19 wherein the nucleic acid is introduced into said cell by liposome transfection.

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Current Assignee
Duke University
Original Assignee
Duke University
Inventors
Smith, James Jefferson, Jantz, Derek, Hellinga, Homme W.
Primary Examiner(s)
RAMIREZ, DELIA M

Application Number

US13/246,355
Publication Number

US 20120028315A1
Time in Patent Office

168 Days
Field of Search

435/6.1, 435/69.1, 435/400, 435/196, 435/320.1, 435/252.3, 435/325
US Class Current

435/18
CPC Class Codes

A61K 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   Antivirals

A61P 43/00   Drugs for specific purposes...

C12N 15/8213   Targeted insertion of genes...

C12N 15/902   using homologous recombination

C12N 15/905   in yeast

C12N 15/907   in mammalian cells

C12N 9/22   Ribonucleases RNAses, DNAse...

Methods of cleaving DNA with rationally-designed meganucleases

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24 Claims

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Methods of cleaving DNA with rationally-designed meganucleases

First Claim

1 Assignment

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Litigations

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Accused Products

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Abstract

52 Citations

24 Claims

Specification

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