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Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination

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  • Behnam Enghiad

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign
    Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign)

  • Chunshuai Huang

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Fang Guo

    (Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign)

  • Guangde Jiang

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Bin Wang

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • S. Kasra Tabatabaei

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Teresa A. Martin

    (Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign)

  • Huimin Zhao

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign
    Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign
    University of Illinois at Urbana-Champaign)

Abstract

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A–F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

Suggested Citation

  • Behnam Enghiad & Chunshuai Huang & Fang Guo & Guangde Jiang & Bin Wang & S. Kasra Tabatabaei & Teresa A. Martin & Huimin Zhao, 2021. "Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination," Nature Communications, Nature, vol. 12(1), pages 1-11, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-21275-4
    DOI: 10.1038/s41467-021-21275-4
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    Cited by:

    1. Yin Liu & Xinyi Liu & Dongyi Wei & Lu Dang & Xiaoran Xu & Shisheng Huang & Liwen Li & Sanyun Wu & Jinxian Wu & Xiaoyan Liu & Wenjun Sun & Wanyu Tao & Yongchang Wei & Xingxu Huang & Kui Li & Xinjie Wan, 2024. "CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    2. 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.
    3. Hengqian Ren & Shravan R. Dommaraju & Chunshuai Huang & Haiyang Cui & Yuwei Pan & Marko Nesic & Lingyang Zhu & David Sarlah & Douglas A. Mitchell & Huimin Zhao, 2023. "Genome mining unveils a class of ribosomal peptides with two amino termini," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    4. Vincent Libis & Logan W. MacIntyre & Rabia Mehmood & Liliana Guerrero & Melinda A. Ternei & Niv Antonovsky & Ján Burian & Zongqiang Wang & Sean F. Brady, 2022. "Multiplexed mobilization and expression of biosynthetic gene clusters," Nature Communications, Nature, vol. 13(1), pages 1-10, December.

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