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

Tianmin Wang, Changge Guan, Jiahui Guo, Bing Liu, Yinan Wu, Zhen Xie, Chong Zhang () and Xin-Hui Xing
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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
Chong Zhang: Tsinghua University
Xin-Hui Xing: Tsinghua University

Nature Communications, 2018, vol. 9, issue 1, 1-15

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-04899-x

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