Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APO-BEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C.G and T.A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

Automated summary

This study developed a range of SpCas9 variants for genome editing in plants, as well as base editors capable of generating conversions at non-canonical PAM sites.