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

ABSTRACT: 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.

SUBMITTER: Zeng X

PROVIDER: S-EPMC10362190 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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

Zeng Xiaoqian X Wang Shuliu S Liang Mindong M Wang Weishan W Jiang Yue Y Xu Fei F Liu Leshi L Yan Hao H Tong Yaojun Y Zhang Lixin L Tan Gao-Yi GY

STAR protocols 20230709 3

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 tr ...[more]

PMID: 37432853

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