Many genes in the bacterial pathogen, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system usingCas9-derived dCas9 for gene knockdown inMultiple genes harbored on either the chromosome or plasmids ofwere efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of(responsible for biofilm formation) or(encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance inphenotypic assays. Furthermore, silencing of the virulence-associated genesorusing this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported forandnull mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening ofgenes with unknown functions.is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, manygenes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported inHere, we systematically optimized the CRISPRi approach for use in, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected byassays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach inand highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen. Yersinia pestis Streptococcus pyogenes Y. pestis Y. pestis hmsH cspB in vitro yscB ail yscB ail Y. pestis Y. pestis Yersinia pestis Y. pestis Y. pestis Y. pestis in vitro Y. pestis IMPORTANCE
