New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen

doi:10.1007/978-1-0716-1720-5_7

. 2022:2377:123-141.

doi: 10.1007/978-1-0716-1720-5_7.

New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen

Xihao Liao¹, Xin-Hui Xing^{1

2}, Chong Zhang^{3

4}

Affiliations

¹ MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China.
² Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China.
³ MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China. chongzhang@tsinghua.edu.cn.
⁴ Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China. chongzhang@tsinghua.edu.cn.

PMID: 34709614
DOI: 10.1007/978-1-0716-1720-5_7

New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen

Xihao Liao et al. Methods Mol Biol. 2022.

. 2022:2377:123-141.

doi: 10.1007/978-1-0716-1720-5_7.

Authors

Xihao Liao¹, Xin-Hui Xing^{1

2}, Chong Zhang^{3

4}

Affiliations

¹ MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China.
² Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China.
³ MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, China. chongzhang@tsinghua.edu.cn.
⁴ Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China. chongzhang@tsinghua.edu.cn.

PMID: 34709614
DOI: 10.1007/978-1-0716-1720-5_7

Abstract

High-throughput genetic screens based on CRISPR/Cas9 technology are powerful tools to genome-wide identify gene function and genotype-phenotype association. Here, we describe a detailed protocol for conducting and evaluating pooled CRISPR screens interfering with gene expression in Escherichia coli. We provide step-by-step instructions for guide RNA library design and construction, genome-scale screening and next-generation sequencing data processing. This tool outperforms transposon sequencing (Tn-seq) with similar library sizes and short gene length. The workflow can be used in follow-up studies implemented in other bacteria systems.

Keywords: CRISPRi; Computational biology; High-throughput screen; Microbial functional genomics; Next-generation sequencing.

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References

1. Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR–Cas9. Nat Rev Genet 16(5):299–311 - DOI
1. Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(1)
1. Koo B-M, Kritikos G, Farelli JD et al (2017) Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Systems 4(3):291–305. e297 - DOI
1. Nichols RJ, Sen S, Choo YJ et al (2011) Phenotypic landscape of a bacterial cell. Cell 144(1):143–156 - DOI
1. Van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10):767 - DOI

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[1] Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR–Cas9. Nat Rev Genet 16(5):299–311 - DOI

[2] Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR–Cas9. Nat Rev Genet 16(5):299–311 - DOI

[3] Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(1)

[4] Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(1)

[5] Koo B-M, Kritikos G, Farelli JD et al (2017) Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Systems 4(3):291–305. e297 - DOI

[6] Koo B-M, Kritikos G, Farelli JD et al (2017) Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Systems 4(3):291–305. e297 - DOI

[7] Nichols RJ, Sen S, Choo YJ et al (2011) Phenotypic landscape of a bacterial cell. Cell 144(1):143–156 - DOI

[8] Nichols RJ, Sen S, Choo YJ et al (2011) Phenotypic landscape of a bacterial cell. Cell 144(1):143–156 - DOI

[9] Van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10):767 - DOI

[10] Van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10):767 - DOI

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New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen

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New Method for Genome-Scale Functional Genomic Study in Bacteria with Superior Performance: CRISPR Interference Screen

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