Improving the Cpf1-mediated base editing system by combining dCas9/dead sgRNA with human APOBEC3A variants

清脆的 生物 基因组编辑 亚基因组mRNA 引导RNA 核酸酶 Cas9 计算生物学 遗传学 索引 基因组工程 基因 基因型 单核苷酸多态性
作者
Meng Lian,Fangbing Chen,Xingyun Huang,Xiaozhu Zhao,Shixue Gou,Nan Li,Qin Jin,Hui Shi,Yanhui Liang,Jingke Xie,Weikai Ge,Zhenpeng Zhuang,Jiaowei Wang,Yinghua Ye,Yi Yang,Kepin Wang,Liangxue Lai,Han Wu
出处
期刊:Journal of Genetics and Genomics [Elsevier BV]
卷期号:48 (1): 92-95 被引量:5
标识
DOI:10.1016/j.jgg.2020.07.010
摘要

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.
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