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Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance

Author

Listed:
  • Tianmin Wang

    (Tsinghua University)

  • Changge Guan

    (Tsinghua University)

  • Jiahui Guo

    (Tsinghua University)

  • Bing Liu

    (Beijing Syngentech Co., Ltd.)

  • Yinan Wu

    (Tsinghua University)

  • Zhen Xie

    (Tsinghua University
    Tsinghua University)

  • Chong Zhang

    (Tsinghua University
    Tsinghua University)

  • Xin-Hui Xing

    (Tsinghua University
    Tsinghua University)

Abstract

To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.

Suggested Citation

  • 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.
  • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-04899-x
    DOI: 10.1038/s41467-018-04899-x
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    Cited by:

    1. Piaopiao Chen & Agnès H. Michel & Jianzhi Zhang, 2022. "Transposon insertional mutagenesis of diverse yeast strains suggests coordinated gene essentiality polymorphisms," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    2. Jiao Liu & Moshi Liu & Tuo Shi & Guannan Sun & Ning Gao & Xiaojia Zhao & Xuan Guo & Xiaomeng Ni & Qianqian Yuan & Jinhui Feng & Zhemin Liu & Yanmei Guo & Jiuzhou Chen & Yu Wang & Ping Zheng & Jibin Su, 2022. "CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    3. 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.
    4. Yichao Han & Wanji Li & Alden Filko & Jingyao Li & Fuzhong Zhang, 2023. "Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    5. Liselot Dewachter & Babette Deckers & Israel Mares-Mejía & Elen Louwagie & Silke Vercauteren & Paul Matthay & Simon Brückner & Anna-Maria Möller & Franz Narberhaus & Sibylle C. Vonesch & Wim Versées &, 2024. "The role of the essential GTPase ObgE in regulating lipopolysaccharide synthesis in Escherichia coli," Nature Communications, Nature, vol. 15(1), pages 1-19, December.

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