An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments

Xiaoqian Zeng; Shuliu Wang; Mindong Liang; Weishan Wang; Yue Jiang; Fei Xu; Leshi Liu; Hao Yan; Yaojun Tong; Lixin Zhang; Gao-Yi Tan

STAR Protocols (Sep 2023)

An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments

Xiaoqian Zeng,
Shuliu Wang,
Mindong Liang,
Weishan Wang,
Yue Jiang,
Fei Xu,
Leshi Liu,
Hao Yan,
Yaojun Tong,
Lixin Zhang,
Gao-Yi Tan

Affiliations

Xiaoqian Zeng: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Shuliu Wang: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Mindong Liang: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Weishan Wang: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Yue Jiang: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Fei Xu: Institute of Pharmaceutical Biotechnology and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
Leshi Liu: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Hao Yan: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Yaojun Tong: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Lixin Zhang: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Gao-Yi Tan: State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; Corresponding author

Journal volume & issue: Vol. 4, no. 3
p. 102435

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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