SpCas9-NG self-targets the sgRNA sequence in plant genome editing

• Published in: Nature plants Vol. 6; no. 3; pp. 197 - 201
• Main Authors: Qin, Ruiying, Li, Juan, Liu, Xiaoshuang, Xu, Rongfang, Yang, Jianbo, Wei, Pengcheng
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-03-2020; Nature Publishing Group
• Subjects: 42/41; 42/70; 631/1647/1513/1967/3196; 631/449/447/2311; Biomedical and Life Sciences; Brief Communication; CRISPR-Associated Protein 9 - genetics; Deoxyribonucleic acid; DNA; DNA, Plant - genetics; Editing; Gene Editing; Gene sequencing; Genome, Plant; Genomes; Life Sciences; Nucleotide sequence; Oryza - genetics; Plant Sciences; Plants, Genetically Modified - genetics; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; RNA, Plant - genetics; Streptococcus pyogenes - genetics; Transgenic plants
• ISSN: 2055-0278; 2055-0278
• DOI: 10.1038/s41477-020-0603-9

Streptococcus pyogenes
Cas9 (SpCas9)-NG recognizes NGN protospacer adjacent motifs and expands the scope of genome-editing tools. In this study, we found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. We further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence.
The widely used SpCas9-NG tool can edit genomic targets, but unfortunately also the guide RNA sequences that are present in the transfer DNA and integrated into the plant genome. The authors propose a strategy to alleviate this self-editing risk.