The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knock-in animal generation via homology directed repair (HDR) is not as efficient as non-homologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knock-in efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18~38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knock-ins or short-nucleotide substitutions by the use of overlapping sgRNAs.

• Su-Cheong Yeom