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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