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

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.