Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand
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ortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand 1,2,3†
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Rong Fan , Zhuangzhuang Chai , Sinian Xing , Kunling Chen , Fengti Qiu , 4 5 1* 2*# 2,3* Tuanyao Chai , Jin-Long Qiu , Zhengbin Zhang , Huawei Zhang & Caixia Gao 1
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Key Laboratory of Agricultural Water Resources, Hebei Laboratory of Agricultural Water-Saving, Center for Agricultural Resources
Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050022, China; State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental
Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; 4 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China; 5 State Key Laboratory of Plant Genomics, Institute of Microbiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, 3
Beijing 100101, China Received April 7, 2020; accepted May 1, 2020; published online June 24, 2020
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants. SpCas9, eSpCas9(1.1), truncated spacer, DSB, nickase, co-editing Citation:
Fan, R., Chai, Z., Xing, S., Chen, K., Qiu, F., Chai, T., Qiu, J.L., Zhang, Z., Zhang, H., and Gao, C. (2020). Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand. Sci China Life Sci 63, https://doi.org/10.1007/s11427-020-1722-0
INTRODUCTION Streptococcus pyogenes Cas9 (SpCas9) is a sequence specific nuclease that interacts with specific DNA targets via sgRNAs (Jiang and Doudna, 2017). The sequence of nucleotides at the 5′ end of the sgRNA (the spacer sequence) base-pairs with the target sequence upstream of the PAM (the protospacer adjacent motif), therefore determining the specificity of SpCas9 (Jiang and Doudna, 2017; Puchta, 2018). †Contributed equally to this work *Corresponding authors (Zhengbin Zhang, email: [email protected]; Huawei Zhang, email: [email protected]; Caixia Gao, email: [email protected]) #Present address: Institute of Advanced Agricultural Science, Peking University, Weifang 261325, China (email: [email protected])
The spacer sequence in SpCas9 sgRNAs usually consists of 20 nucleotides
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