Method for producing genetically-modified cells with rationally-designed meganucleases with altered sequence specificity

US 8,377,674 B2
Filed: 06/25/2012
Issued: 02/19/2013
Est. Priority Date: 10/18/2005
Status: Active Grant

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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 nucleic acid 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 one 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 at least one 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, 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;

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 nucleic acid in the chromosome and the target nucleic acid 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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1. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target nucleic acid 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 one 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 at least one 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, 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;
  
  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 nucleic acid in the chromosome and the target nucleic acid 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)
- - 2. The method of claim 1, further comprising:
    - introducing into the cell a second nucleic acid comprising a nucleic acid of interest;
      
      whereby the target nucleic acid is modified by insertion of the nucleic acid of interest into the chromosome.
  - 3. The method of claim 2, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the nucleic acid of interest is inserted into the chromosome by homologous recombination.
  - 4. The method of claim 2, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the nucleic acid of interest is inserted into the chromosome by non-homologous end-joining.
  - 5. The method of claim 1, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.

6. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target nucleic acid 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 one 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 at least one 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, 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;
  
  wherein the polypeptide further comprises a second amino acid modification affecting heterodimer formation corresponding to a substitution in the polypeptide of SEQ ID NO;
  
  1 selected from the group consisting of;
  
  K7D, K7E, E8R, K57D, K57E, E61K, E61R, K96D and K96E; and
  
  wherein the recombinant meganuclease cleaves a site in the target nucleic acid in the chromosome and the target nucleic acid 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 (7, 8, 9, 10)
- - 7. The method of claim 6, further comprising:
    - introducing into the cell a second nucleic acid comprising a nucleic acid of interest;
      
      whereby the target nucleic acid is modified by insertion of the nucleic acid of interest into the chromosome.
  - 8. The method of claim 7, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the nucleic acid of interest is inserted into the chromosome by homologous recombination.
  - 9. The method of claim 7, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the nucleic acid of interest is inserted into the chromosome by non-homologous end-joining.
  - 10. The method of claim 6, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.

11. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target nucleic acid 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 one 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 nucleic acid 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;
- View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25)
- - 12. The method of claim 11, further comprising:
    - introducing into the cell a second nucleic acid comprising a nucleic acid of interest;
      
      whereby the target nucleic acid is modified by insertion of the nucleic acid of interest into the chromosome.
  - 13. The method of claim 12, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the nucleic acid of interest is inserted at into the chromosome by homologous recombination.
  - 14. The method of claim 12, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the nucleic acid of interest is inserted into the chromosome by non-homologous end-joining.
  - 15. The method of claim 11, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.
  - 16. The method of claim 11, wherein the nucleotide modification corresponds to position −
    - 1 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ II) NO;
      
      4 has been altered to an A, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1selected from the group consisting of D75C, D75L, D75Y, K139Y, T46A T46C, T46G and R70A;
      
      (ii) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1selected from the group consisting of T46E, T46D, R70E, D75E, T46G and R70A; and
      
      iii) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of R70C, R70Q, D75H, D75Q, D75Y K139H, T46H, T46Q, and T46G.
  - 17. The method of claim 11, wherein the nucleotide modification corresponds to position 31 3 of the I-CreI recognition sequence of SEQ ID NO:
    - 4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      3 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of 124C; and
      
      (ii) if the nucleotide corresponding to position −
      
      3 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of I24K and I24R.
  - 18. The method of claim 11 wherein the nucleotide modification corresponds to position −
    - 4 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1selected from the group consisting of I77E, I775, Q26K and Q26S;
      
      (ii) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of I77R, Q26E and Q26S; and
      
      (iii) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of Q26S.
  - 19. The method of claim 11, wherein the nucleotide modification corresponds to position −
    - 5 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 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 at least one 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 of SEQ ID NO;
      
      4 has been altered to an A, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of A42Q, Y66M, and Y66K; and
      
      (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 at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of Y66M and Y66K.
  - 20. The method of claim 11, wherein the nucleotide modification corresponds to position −
    - 6 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S40C, S40I, S40V, S79A, S79C, S79I, S79V and K28H;
      
      (ii) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of K28S; and
      
      (iii) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S40R and K28S.
  - 21. The method of claim 11, wherein the nucleotide modification corresponds to position −
    - 8 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein if the nucleotide corresponding to position −
      
      8 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of S32R.
  - 22. The method of claim 11, wherein the nucleotide modification corresponds to position −
    - 9 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      9 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S32N, S32H S32Q and S32T;
      
      (ii) if the nucleotide corresponding to position −
      
      9 of the I-CreI. meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S32L, S32V, S32C, S32D, S32I, S32N, S32H, S32Q and S32T; and
      
      (iii) if the nucleotide corresponding to position −
      
      9 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the at least one specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S32N, S32H, S32Q and S32T.
  - 24. The method of claim 11, wherein the recombinant meganuclease further comprises at least a second specificity-altering amino acid modification affecting specificity at position −
    - 2 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 corresponding to a substitution in SEQ ID NO;
      
      1, wherein said substitution is selected form group consisting of Q44A, Q44C, Q44D, Q44E, Q44I, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, R70D, R70E, R70H and R70Q.
  - 25. The method of claim 11, wherein the recombinant meganuclease further comprises at least a second specificity-altering amino acid modification affecting specificity at position −
    - 7 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 corresponding to a substitution in SEQ ID NO;
      
      1, wherein said substitution is selected form group consisting of N30E, N30K, N30Q, N30R, Q38C, Q38E, Q38H, Q38I, Q38K, Q38L, Q38N and Q38R.

23. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target nucleic acid 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 target nucleic acid 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 ;

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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

US13/532,190
Publication Number

US 20120266266A1
Time in Patent Office

239 Days
Field of Search

435/18, 435/6.1, 435/196, 435/69.1, 530/350
US Class Current

435/196
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...

Method for producing genetically-modified cells with rationally-designed meganucleases with altered sequence specificity

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Method for producing genetically-modified cells with rationally-designed meganucleases with altered sequence specificity

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