Construction of an arrayed CRISPRi library as a resource for essential gene function studies in Streptococcus mutans

Arrayed mutant libraries are important resources that have advanced our understanding of bacterial genetics. Until recently, essential genes which are necessary for bacterial survival, and constitute similar to 10% of bacterial genes, were not included in arrayed library resources. However, recent advances in clustered regularly interspaced short palindromic repeats interference (CRISPRi) have made it possible to study essential genes with increasing throughput. Here, we constructed an arrayed CRISPRi library in the dental caries pathogen Streptococcus mutans, known as SNAP (Streptococcus mutans arrayed CRISPRi). In this library, each strain contains a short guide RNA which selectively targets a single essential gene, and this is controlled through xylose induction. In total, the library can selectively repress >250 essential and growth-supporting genes. Initial characterization showed that SNAP strains grow as expected, and initial proof-of-concept experiments displayed the utility of the library. We anticipate that this library will be of benefit to the research community by allowing for high-throughput characterization of S. mutans essential genes. IMPORTANCE The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as Streptococcus mutans arrayed CRISPRi(SNAP), as a resource to study >250 essential and growth-supporting genes in Streptococcus mutans. SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.