Altered Recombinases for Genome Modification
OwnerChristopher R. Sclimentiinventor
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Abstract
The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.
Claims17
1-22. (canceled)
An altered, unidirectional site-specific bacteriophage integrase comprising an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, with integrase activity.
A method of site-specifically integrating a nucleic acid into a genome of a cell, said method comprising:
- a. introducing a targeting vector comprising said nucleic acid and a vector attachment site into a cell;
- b. introducing an altered, unidirectional site-specific bacteriophage integrase into said cell;
- and
- c. maintaining said cell under conditions sufficient for said vector to integrate into said genome of said cell by a recombination event mediated by said altered unidirectional site-specific bacteriophage integrase, wherein said unidirectional site-specific bacteriophage integrase comprises an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, and wherein said integrase has integrase activity.
A method of site-specifically integrating a nucleic acid into a genome of a cell, said method comprising:
- a. introducing a targeting vector comprising said nucleic acid and a vector attachment site into a cell;
- b. introducing an altered, unidirectional site-specific bacteriophage integrase into said cell;
- and
- c. maintaining said cell under conditions sufficient for said vector to integrate into said genome of said cell by a recombination event mediated by said altered unidirectional site-specific bacteriophage integrase, wherein the nucleic acid is integrated into the genome at a site comprising a sequence having at least 40% sequence identity to the approximately 40 base pair nucleic acid sequence surrounding the core TTG nucleic acid sequence of SEQ ID NO:33 or SEQ ID NO:34.
An altered, unidirectional site-specific bacteriophage integrase that is capable of integrating a nucleic acid into a genome of a eukaryotic cell at a site comprising a sequence having at least 40% sequence identity to SEQ ID NO:33 or SEQ ID NO:34.
A kit for use in integrating a nucleic acid into a genome of a cell of a multicellular organism, said kit comprising:
- (a) a targeting vector comprising said nucleic acid and a vector attachment site;
- (b) an altered, unidirectional site-specific bacteriophage integrase or a nucleic acid encoding the same, wherein said unidirectional site-specific bacteriophage integrase has at least 80% sequence identity to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, wherein said integrase has integrase activity.
Dependencies are inferred from the claim text.