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CRISPRi-seq for genome-wide fitness quantification in bacteria

Nature Protocols volume 17, pages 252–281 (2022)Cite this article

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Abstract

CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2–3 weeks.

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Fig. 1: Growth curves of individual CRISPRi strains indicate essentiality of sgRNA targets.
Fig. 2: Overview of the CRISPRi-seq workflow.
Fig. 3: Construction of the pooled CRISPRi library.
Fig. 4: Workflow of the experimental CRISPRi-seq screening steps.
Fig. 5: Example outcomes of differential sgRNA enrichment analyses.

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Data availability

Source data are provided with this paper.

Code availability

Source code for the sgRNA design and evaluation pipelines, as well as for the naïve power analysis and corresponding data, can be found at https://github.com/veeninglab/CRISPRi-seq. 2FAST2Q code, files and executables can be found at https://github.com/veeninglab/2FAST2Q, https://pypi.org/project/fast2q/ or https://doi.org/10.5281/zenodo.5079789. (Off-)target binding site tables from Liu et al.11 can be found through our website: https://www.veeninglab.com/crispri-seq.

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Acknowledgements

Work in the Veening lab is supported by the Swiss National Science Foundation (SNSF) (project grants 31003A_172861 and 310030_192517), SNSF JPIAMR grant (40AR40_185533), SNSF NCCR ‘AntiResist’ (51NF40_180541) and ERC consolidator grant 771534-PneumoCaTChER.

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Author notes

  1. These authors contributed equally: Vincent de Bakker, Xue Liu.

Authors and Affiliations

  1. Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

    Vincent de Bakker, Xue Liu, Afonso M. Bravo & Jan-Willem Veening

  2. Guangdong Key Laboratory for Genome Stability and Human Disease Prevention, Department of Pharmacology, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, China

    Xue Liu

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Contributions

X.L. developed the experimental procedures. V.d.B. developed the computational/analysis procedures. A.M.B. developed 2FAST2Q and the Python code for the computational pipelines. J.W.V. conceptualized this study. V.d.B. and X.L. wrote the original manuscript draft with input from A.M.B. V.d.B., X.L., A.M.B. and J.W.V. revised and edited to obtain the final manuscript.

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Correspondence to Jan-Willem Veening.

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Key references using this protocol

Liu, X. et al. Cell Host Microbe 29, 107–120.e6 (2021): https://doi.org/10.1016/j.chom.2020.10.001

Liu, X. et al. Mol. Syst. Biol. 13, 931 (2017): https://doi.org/10.15252/msb.20167449

Key data used in this protocol

Liu, X. et al. Cell Host Microbe 29, 107–120.e6 (2021): https://doi.org/10.1016/j.chom.2020.10.001

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Supplementary Methods.

Supplementary Software

Custom Illumina MiniSeq sequencing recipes with 54 dark cycles for both Mid and High Output Kits

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de Bakker, V., Liu, X., Bravo, A.M. et al. CRISPRi-seq for genome-wide fitness quantification in bacteria. Nat Protoc 17, 252–281 (2022). https://doi.org/10.1038/s41596-021-00639-6

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