Feasibility of a Conditional Knockout System for Based on CRISPR Interference.

is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of with an exogenous plasmid, genetic manipulation of remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of to inducibly, and reversibly, repress gene expression in . CRISPRi has been developed and used successfully in a variety of bacterial organisms including and . I first describe the creation of a single plasmid system for CRISPRi in , targeted to a non-essential gene, , that expresses a dispensable inclusion membrane protein. Control transformations of serovar L2 with plasmids encoding only the dCas9, under the control of an inducible promoter, or only the guide RNA (gRNA) targeted to the 5' UTR of , expressed constitutively, failed to prevent expression of IncA. Importantly, expression of dCas9 alone did not have a deleterious effect on chlamydiae. Transformation of with a plasmid combining the dCas9 and a gRNA targeting and induction of expression of the dCas9 resulted in the reversible inhibition of IncA expression. Consequently, conditional knockout mediated by CRISPRi is feasible in . Potential improvements and experimental concerns in using the system are also discussed.