Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist’s toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.

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