AUTHOR=Jeong Song Hee , Lee Ho Joung , Lee Sang Jun TITLE=Use of paired Cas9-NG nickase and truncated sgRNAs for single-nucleotide microbial genome editing JOURNAL=Frontiers in Genome Editing VOLUME=6 YEAR=2024 URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2024.1471720 DOI=10.3389/fgeed.2024.1471720 ISSN=2673-3439 ABSTRACT=

The paired nickases approach, which utilizes clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) nickase and dual guide RNA, has the advantage of reducing off-target effects by being able to double the target sequence. In this study, our research utilized the Cas9-NG nickase variant to minimize PAM sequence constraints, enabling the generation of paired nicks at desired genomic loci. We performed a systematic investigation into the formation sites for double nicks and the design of donor DNA within a bacterial model system. Although we successfully identified the conditions necessary for the effective formation of double nicks in vivo, achieving single-nucleotide level editing directly at the target sites in the genome proved challenging. Nonetheless, our experiments revealed that efficient editing at the single-nucleotide level was achievable on target DNA sequences that are hybridized with 5′-end-truncated dual single-guide RNAs (sgRNAs). Our findings contribute to a deeper understanding of the paired nickases approach, offering a single-mismatch intolerance design strategy for accurate nucleotide editing. This strategy not only enhances the precision of genome editing but also marks a significant step forward in the development of nickase-derived genome editing technologies.