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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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
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author Zeng, Xiaoqian
Wang, Shuliu
Liang, Mindong
Wang, Weishan
Jiang, Yue
Xu, Fei
Liu, Leshi
Yan, Hao
Tong, Yaojun
Zhang, Lixin
Tan, Gao-Yi
author_facet Zeng, Xiaoqian
Wang, Shuliu
Liang, Mindong
Wang, Weishan
Jiang, Yue
Xu, Fei
Liu, Leshi
Yan, Hao
Tong, Yaojun
Zhang, Lixin
Tan, Gao-Yi
author_sort Zeng, Xiaoqian
collection PubMed
description 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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spelling pubmed-103621902023-07-23 An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments Zeng, Xiaoqian Wang, Shuliu Liang, Mindong Wang, Weishan Jiang, Yue Xu, Fei Liu, Leshi Yan, Hao Tong, Yaojun Zhang, Lixin Tan, Gao-Yi STAR Protoc Protocol 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) Elsevier 2023-07-09 /pmc/articles/PMC10362190/ /pubmed/37432853 http://dx.doi.org/10.1016/j.xpro.2023.102435 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Zeng, Xiaoqian
Wang, Shuliu
Liang, Mindong
Wang, Weishan
Jiang, Yue
Xu, Fei
Liu, Leshi
Yan, Hao
Tong, Yaojun
Zhang, Lixin
Tan, Gao-Yi
An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title_full An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title_fullStr An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title_full_unstemmed An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title_short An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments
title_sort in vitro crispr-cas12a-mediated protocol for direct cloning of large dna fragments
topic Protocol
url 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
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