J Integr Plant Biol. ›› 2021, Vol. 63 ›› Issue (9): 1611-1619.DOI: 10.1111/jipb.13164

• Research Articles • Previous Articles     Next Articles

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

Taoli Liu1,2†, Dongchang Zeng1,2,3†, Zhiye Zheng2, Zhansheng Lin1,2, Yang Xue1,2, Tie Li1,2, Xianrong Xie1,2, Genglu Ma1,2, Yao‐Guang Liu1,2,3* and Qinlong Zhu1,2,3*   

  1. 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro‐Bioresources, Guangzhou 510642, China
    2 College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
    3 Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China

    These authors contributed equally to this work.
    *Correspondences: Yao‐Guang Liu (ygliu@scau.edu.cn); Qinlong Zhu (zhuql@scau.edu.cn, Dr. Zhu is fully responsible for the distribution of the materials associated with this article)
  • Received:2021-07-04 Accepted:2021-08-16 Online:2021-08-19 Published:2021-09-01

Abstract: 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 (C1–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.

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