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

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...

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Detalles Bibliográficos
Autores principales: Zeng, Xiaoqian, Wang, Shuliu, Liang, Mindong, Wang, Weishan, Jiang, Yue, Xu, Fei, Liu, Leshi, Yan, Hao, Tong, Yaojun, Zhang, Lixin, Tan, Gao-Yi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10362190/
https://www.ncbi.nlm.nih.gov/pubmed/37432853
http://dx.doi.org/10.1016/j.xpro.2023.102435
Descripción
Sumario: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)

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