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US 2012/0070877 A1Published application
Rationally designed meganucleases with altered sequence specificity and dna-binding affinity
AssigneeDuke University
InventorsJames J. Smith, Derek Jantz, Homme W. Hellinga
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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.
Claims13
A I-CreI meganuclease variant, comprising the following substitutions:
- K28Q, Q38K and S40R of the wild-type I-CreI (SEQ ID NO:
- 1), said variant being able to cleave a DNA target sequence consisting of a mutant I-CreI site wherein at least the nucleotide A in position -7 and/or -6, and/or the nucleotide T in position +6 and/or +7 has been replaced with a different nucleotide.
A I-CreI meganuclease variant having a modified cleavage specificity, wherein
- (a) at least one of the amino acids K28 and/or S40 from the 131132 hairpin of the wild-type I-CreI (SEQ ID NO:
- 1) has been substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, K, N, P, Q, R, S, T, L, V, W and Y and optionally
- (b) at least one of the amino acids N30, Y33 and Q38 from the 131132 hairpin of the wild-type I-CreI (SEQ ID NO:
- 1) has been substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, K, N, P, Q, R, S, T, L, V, W and Y;
- and
- wherein the I-CreI variant is able to cleave a DNA target sequence consisting of a mutant I-CreI site wherein at least the nucleotide doublet AA in positions -7 and -6 and/or the nucleotide doublet TT in positions +6 to +7 has been replaced with a different nucleotide doublet.
3 more elements
Dependencies are inferred from the claim text.