Patent ID: 7314712

Claim:
A method for introducing a mutation into a target double stranded nucleic acid sequence in a cell, wherein the double stranded nucleic acid sequence comprises a first and a second strand, the method comprising: introducing a double-stranded nucleic acid cassette into a target nucleic acid sequence at an insertion point, wherein the cassette is a reporter double strand break cassette (RE-DSB-cassette) and comprises: a first portion homologous to a nucleic acid sequence on a first side of the insertion point; a second portion homologous to a second nucleic acid sequence on a second side of the insertion point; a nucleic acid sequence encoding a reporter located between the first portion and the second portion; a nucleic acid sequence comprising a double-strand break recognition site; a nucleic acid encoding a double-strand break enzyme that recognizes the double-strand break recognition site and generates a double-strand break at or near the double-strand break recognition site; and an inducible promoter, operably connected with the nucleic acid encoding the double-strand break enzyme; transforming the cell with a first oligonucleotide comprising: a nucleic acid sequence homologous to one strand (the chosen strand) of the target nucleic acid sequence at a position on the first side of the insertion point; and a nucleic acid sequence homologous to the same strand of the target nucleic acid sequence at a position on the second side of the insertion point, and comprising at least one nucleotide that differs from the chosen strand of the target nucleic acid sequence; and selecting for complete removal of the nucleic acid sequence encoding the reporter gene, wherein complete removal of the nucleic acid sequence encoding the reporter gene indicates integration of the oligonucleotide sequence comprising the at least one nucleotide that differs from the target nucleic acid sequence.