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Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

Author

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  • Wenjun Jiang

    (School of Life Sciences, Center for Synthetic and Systems Biology, MOE Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University)

  • Xuejin Zhao

    (CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences)

  • Tslil Gabrieli

    (Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University)

  • Chunbo Lou

    (CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences)

  • Yuval Ebenstein

    (Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University)

  • Ting F. Zhu

    (School of Life Sciences, Center for Synthetic and Systems Biology, MOE Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University)

Abstract

The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.

Suggested Citation

  • Wenjun Jiang & Xuejin Zhao & Tslil Gabrieli & Chunbo Lou & Yuval Ebenstein & Ting F. Zhu, 2015. "Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters," Nature Communications, Nature, vol. 6(1), pages 1-8, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms9101
    DOI: 10.1038/ncomms9101
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    Cited by:

    1. Alessandro L. V. Coradini & Christopher Ne Ville & Zachary A. Krieger & Joshua Roemer & Cara Hull & Shawn Yang & Daniel T. Lusk & Ian M. Ehrenreich, 2023. "Building synthetic chromosomes from natural DNA," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    2. Taotao Li & Duo Du & Dandan Zhang & Yicheng Lin & Jiakang Ma & Mengyu Zhou & Weida Meng & Zelin Jin & Ziqiang Chen & Haozhe Yuan & Jue Wang & Shulong Dong & Shaoyang Sun & Wenjing Ye & Bosen Li & Houb, 2023. "CRISPR-based targeted haplotype-resolved assembly of a megabase region," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    3. Hideto Mori & Nozomu Yachie, 2022. "A framework to efficiently describe and share reproducible DNA materials and construction protocols," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
    4. Qun Zhou & Yatong Zhao & Changqiang Ke & Haojun Wang & Sheng Gao & Hui Li & Yan Zhang & Yang Ye & Yunzi Luo, 2024. "Repurposing endogenous type I-E CRISPR-Cas systems for natural product discovery in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    5. Yuqian Tang & Debin Qin & Zhexian Tian & Wenxi Chen & Yuanxi Ma & Jilong Wang & Jianguo Yang & Dalai Yan & Ray Dixon & Yi-Ping Wang, 2023. "Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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