The ScCas9++ variant expands the CRISPR toolbox for genome editing in plants.

The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9+ and ScCas9++ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9+ and ScCas9++ and a nickase variant ScCas9n++ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9++ has higher editing efficiency than ScCas9 and ScCas9+ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++ . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17 ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9++ represents a crucial new tool for improving plant editing.