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Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs

Author

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  • Jae Sung Cho

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    Massachusetts Institute of Technology)

  • Dongsoo Yang

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    Korea University)

  • Cindy Pricilia Surya Prabowo

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST)

  • Mohammad Rifqi Ghiffary

    (Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    Systems Biology and Medicine Laboratory, Department of Chemical and Biomolecular Engineering (BK21 four), KAIST)

  • Taehee Han

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST)

  • Kyeong Rok Choi

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST)

  • Cheon Woo Moon

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST)

  • Hengrui Zhou

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST)

  • Jae Yong Ryu

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    Duksung Women’s University)

  • Hyun Uk Kim

    (Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    Systems Biology and Medicine Laboratory, Department of Chemical and Biomolecular Engineering (BK21 four), KAIST
    BioProcess Engineering Research Center and BioInformatics Research Center, KAIST)

  • Sang Yup Lee

    (Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
    Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
    BioProcess Engineering Research Center and BioInformatics Research Center, KAIST)

Abstract

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Suggested Citation

  • Jae Sung Cho & Dongsoo Yang & Cindy Pricilia Surya Prabowo & Mohammad Rifqi Ghiffary & Taehee Han & Kyeong Rok Choi & Cheon Woo Moon & Hengrui Zhou & Jae Yong Ryu & Hyun Uk Kim & Sang Yup Lee, 2023. "Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38119-y
    DOI: 10.1038/s41467-023-38119-y
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    References listed on IDEAS

    as
    1. Tianmin Wang & Changge Guan & Jiahui Guo & Bing Liu & Yinan Wu & Zhen Xie & Chong Zhang & Xin-Hui Xing, 2018. "Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance," Nature Communications, Nature, vol. 9(1), pages 1-15, December.
    2. Fernando P. Canale & Camilla Basso & Gaia Antonini & Michela Perotti & Ning Li & Anna Sokolovska & Julia Neumann & Michael J. James & Stefania Geiger & Wenjie Jin & Jean-Philippe Theurillat & Kip A. W, 2021. "Metabolic modulation of tumours with engineered bacteria for immunotherapy," Nature, Nature, vol. 598(7882), pages 662-666, October.
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