Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice

The Streptococcus canis Cas9 protein (ScCas9) recognizes the NNG protospacer adjacent motif (PAM), offering a wider range of targets than that offered by the commonly used S. pyogenes Cas9 protein (SpCas9). However, both ScCas9 and its evolved Sc++ variant still exhibit low genome editing efficiency in plants, particularly at the less preferred NTG and NCG PAM targets. In this study, a chimeric SpcRN++ variant is engineered by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the R221K/N394K mutations, and retaining the positively charged loop of S. anginosus Cas9. The SpcRN++ variant exhibits a higher genome editing capacity and wider target range than the Sc++ variant in rice protoplasts and stable transgenic plants. Further evidence indicates that nSpcRN++-based A3A/Y130F and TadA8e exhibit enhanced cytosine and adenine editing efficiency in plants. Finally, herbicide-resistant rice germplasms are produced by targeting the OsACC gene using nSpcRN++-based adenine base editors. These results demonstrate that SpcRN++ is a powerful tool for genome editing in plants, and this integrative protein engineering strategy holds promise for engineering other Cas9 proteins.