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

Behnam Enghiad, Chunshuai Huang, Fang Guo, Guangde Jiang, Bin Wang, S. Kasra Tabatabaei, Teresa A. Martin and Huimin Zhao ()
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Behnam Enghiad: Carl R. Woese Institute for Genomic Biology, 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

Nature Communications, 2021, vol. 12, issue 1, 1-11

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-21275-4

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