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–C
17) 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.
相似文献