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‐protospac...

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Bibliographic Details
Published in:	Journal of integrative plant biology Vol. 63; no. 9; pp. 1611 - 1619
Main Authors:	Liu, Taoli, Zeng, Dongchang, Zheng, Zhiye, Lin, Zhansheng, Xue, Yang, Li, Tie, Xie, Xianrong, Ma, Genglu, Liu, Yao‐Guang, Zhu, Qinlong
Format:	Journal Article
Language:	English
Published:	China (Republic : 1949- ) Wiley Subscription Services, Inc 01-09-2021 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Guangzhou 510642,China College of Life Sciences,South China Agricultural University,Guangzhou 510642,China%State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Guangzhou 510642,China Guangdong Laboratory for Lingnan Modern Agriculture,Guangzhou 510642,China%College of Life Sciences,South China Agricultural University,Guangzhou 510642,China College of Life Sciences,South China Agricultural University,Guangzhou 510642,China
Subjects:	base editing CRISPR CRISPR-Associated Protein 9 - genetics CRISPR-Cas Systems Cytidine deaminase Editing Efficiency Gene Editing - methods genome editing Genomes Germplasm Multiplexing Oryza - genetics Oryza sativa Proteins Rice ScCas9 Streptococcus - genetics CRISPR ScCas9 genome editing rice base editing
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Summary:	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. The nuclease ScCas9 recognizes the protospacer‐adjacent motif NNG, but its editing activity is low in plants. However, the engineered evolved ScCas9++ has higher editing efficiency than other ScCas9 variants in rice and its nickase variant ScCas9n++ has improved potential for developing base‐editing tools with expanded target scope in plants.
Bibliography:	These authors contributed equally to this work. Lanqin Xia, Institute of Crop Sciences, CAAS, China Edited by ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23
ISSN:	1672-9072 1744-7909
DOI:	10.1111/jipb.13164