文献类型：期刊文献

英文题名：An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments

作者：Zeng, Xiaoqian[1,2];Wang, Shuliu[1,2];Liang, Mindong[1,2];Wang, Weishan[3];Jiang, Yue[1,2];Xu, Fei[4,5];Liu, Leshi[1,2];Yan, Hao[3];Tong, Yaojun[6,7];Zhang, Lixin[1,2];Tan, Gao-Yi[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Sch Biotechnol, Shanghai 200237, Peoples R China;[3]Chinese Acad Sci, State Key Lab Microbial Resources, Inst Microbiol, Beijing 100101, Peoples R China;[4]Zhejiang Univ, Affiliated Hosp 2, Inst Pharmaceut Biotechnol, Sch Med, Hangzhou 310058, Peoples R China;[5]Zhejiang Univ, Dept Gastroenterol, Affiliated Hosp 2, Sch Med, Hangzhou 310058, Peoples R China;[6]Shanghai Jiao Tong Univ, State Key Lab Microbial Metab, Joint Int Res Lab Metab & Dev Sci, Shanghai 200240, Peoples R China;[7]Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China

年份：2023

卷号：4

期号：3

外文期刊名：STAR PROTOCOLS

收录：WOS:【ESCI(收录号:WOS:001058642600001)】；

基金：This work was financially supported by National Key Research and Development Program of China [2020YFA0907800, 2021YFC2100600, and 2020YFA0907304] , the 111 Project (B18022) , Fundamental Research Funds for the Central Universities [22221818014] , and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

语种：英文

摘要：Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al.1