Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity

  • US 8,021,867 B2
  • Filed: 10/18/2006
  • Issued: 09/20/2011
  • Est. Priority Date: 10/18/2005
  • Status: Active Grant
First Claim
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1. A recombinant meganuclease having altered cleavage specificity relative to the wild-type I-CreI meganuclease of SEQ ID NO:

  • 1, comprising a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

    1, which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

    1 by;

    (a) 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

    (b) at least a second specificity-altering amino acid modification at a position corresponding to position 26 or 77 of SEQ ID NO;

    1, wherein said second specificity-altering amino acid modification is a substitution selected from the group consisting of Q26E, Q26K, Q26S, I77E, I77Q, I77R and I77S; and

    (c) at least a third amino acid modification affecting heterodimer formation selected from the group consisting of K7D, K7E, E8K, E8R, K57D, K57E, E61K, E61R, K96D and K96E.

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