Abstract: Described herein are methods of enhancing chromosomal homologous recombination to stimulate a loss of heterozygosity at a gene locus of interest in a living cell. These methods are driven by an enhancer component and a target-specific endonuclease component and proceed through a mechanism whereby: exogenous donor DNA that is homologous to the gene locus of interest is not introduced into the living cell; the desired allele of the gene locus of interest remains uncleaved; and the undesired allele is either uncleaved, cleaved at a single location, or cleaved at multiple locations. These methods have numerous applications, including the repair of risk alleles for disease prevention, the correction of heterozygous mutations in dividing cells, the design of cancer therapeutics, and the design of novel gene-drive strategies.

Abstract: An object of the present invention is to provide a method of conveniently producing a genetically modified non-human mammal with high efficiency using a CRISPR-Cas9 system and particularly a production method whereby gene knock-in can be achieved with high efficiency regardless of the gene size. The method of producing a genetically modified non-human mammal comprises introducing a Cas9 protein, a crRNA fragment comprising a nucleotide sequence complementary to a target DNA region, and a tracrRNA fragment into a non-human mammalian oocyte to genetically modify the target DNA.

Abstract: Described herein are methods of enhancing chromosomal homologous recombination to stimulate a loss of heterozygosity at a gene locus of interest in a living cell. These methods are driven by an enhancer component and a target-specific endonuclease component and proceed through a mechanism whereby: exogenous donor DNA that is homologous to the gene locus of interest is not introduced into the living cell; the desired allele of the gene locus of interest remains uncleaved; and the undesired allele is either uncleaved, cleaved at a single location, or cleaved at multiple locations. These methods have numerous applications, including the repair of risk alleles for disease prevention, the correction of heterozygous mutations in dividing cells, the design of cancer therapeutics, and the design of novel gene-drive strategies.

Abstract: The present inventors tried to prepare a knock-in animal using a genome editing system containing a nuclease in the form of protein and a DNA-targeting RNA and using a single-stranded DNA as a donor, and found that it is possible to obtain a cell in which the donor DNA has been knocked in with extremely high efficiency.

Abstract: An object of the present invention is to provide a method of conveniently producing a genetically modified non-human mammal with high efficiency using a CRISPR-Cas9 system and particularly a production method whereby gene knock-in can be achieved with high efficiency regardless of the gene size. The method of producing a genetically modified non-human mammal comprises introducing a Cas9 protein, a crRNA fragment comprising a nucleotide sequence complementary to a target DNA region, and a tracrRNA fragment into a non-human mammalian oocyte to genetically modify the target DNA.