The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous 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 knockin 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 knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.
Bibliographical noteFunding Information:
This work was supported by grants from the National Research Foundation (Nos. 2015R1C1A1A01051949, 2015R1A2A1A01003845), the Korea Food and Drug Administration (14182KFDA978), and the Yonsei University Yonsei-SNU collaborative research fund of 2014. This work was supported by grants from the National Research Foundation (Nos. 2015R1C1A1A01051949, 2015R1A2A1A01003845, and 2017R1A4A1015328), the Korea Food and Drug Administration (14182KFDA978), and the Yonsei University Yonsei-SNU collaborative research fund of 2014.
All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Clinical Biochemistry