Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency

Abstract

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. A method to improve the efficiency of repairing double-stranded DNA breaks facilitates the production of animals with edited genomes. The CRISPR/Cas9 system has been revolutionising biomedical research as it allows the insertion or deletion of any DNA sequence at specific sites by making double-strand DNA breaks. However, the insertion of precise genetic changes is limited by the relatively low efficiency of the repair mechanism, which requires a template DNA molecule to promote error-free repair of the DNA strands. A team of South Korean scientists led by Su Cheong Yeom at Seoul National University and Han Woong Lee at Yonsei University show that delivering multiple overlapping single-guide RNAs (that share at least five base pairs of the target sequence) as a DNA template significantly improves the repair and thus the production of mice with a targeted gene insertion.