机构：[1]Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China[2]School of Life Science and Technology, Shanghai Tech University, Shanghai, China[3]University of Chinese Academy of Sciences, Beijing, China[4]State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China[5]Sun Yat-sen University Cancer Center, Guangzhou, China中山大学肿瘤防治中心[6]Shanghai Tolo Biotechnology Company Limited, Shanghai, China
The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.
基金：
Chinese Academy of Sciences [XDB19040200]; Youth Innovation Promotion Association CAS; National Natural Science Foundation of China [31430004, 31421061]
语种：
外文
被引次数：
WOS：
PubmedID：
中科院(CAS)分区：
出版当年[2017]版：
无
最新[2023]版：
大类|1 区生物学
小类|2 区细胞生物学
第一作者：
第一作者机构：[1]Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China[2]School of Life Science and Technology, Shanghai Tech University, Shanghai, China[3]University of Chinese Academy of Sciences, Beijing, China
共同第一作者：
通讯作者：
通讯机构：[1]Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
推荐引用方式(GB/T 7714)：
Xiaochun Zhang,Jingman Wang,Qiuxiang Cheng,et al.Multiplex gene regulation by CRISPR-ddCpf1[J].CELL DISCOVERY.2017,3:-.doi:10.1038/celldisc.2017.18.
