Zixu Zhu
Guanwen Liu
Chao Sun
Dexing Lin
Chenxiao Xue
Shengnan Li
Dandan Zhang
Caixia Gao
Yanpeng Wang
Jin-Long Qiu
a 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;
b College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;
c State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
d CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing 100049, China
Funds: This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020101 and XDA24020310), the National Natural Science Foundation of China (31672015, 31788103) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003).
Received Date: 2021-02-14
Accepted Date:2021-04-11
Rev Recd Date:2021-04-03
Publish Date:2021-06-20
Abstract
Abstract
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We develop two variants, MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5′-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.Keywords: MAD7 nuclease,
CRISPR-Cas12a,
Plant genome editing,
Royalty-free,
Commercial use
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