CRISPR Interference to Inducibly Repress Gene Expression in Chlamydia trachomatis
The ability to inducibly repress gene expression is critical to the study of organisms, like Chlamydia, with reduced genomes wherein the majority of genes are likely to be essential. We recently described the feasibility of a CRISPR interference system to inducibly repress gene expression in Chlamydia trachomatis. However, the initial system suffered from some drawbacks, primarily leaky expression of the anhydrotetracycline (aTc) inducible dCas9 ortholog and plasmid instability, that prevented population-wide studies (e.g. transcript analyses) of the effects of knockdown. Here, we describe various modifications to the original system that have allowed us to measure gene expression changes within a transformed population of C. trachomatis serovar L2. These modifications include (i) a change in the vector backbone, (ii) the introduction of a weaker ribosome binding site driving dCas9 translation, and (iii) the addition of a degradation tag to the dCas9 itself. With these changes, we demonstrate the ability to inducibly repress a target gene sequence as measured by the absence of protein by immunofluorescence analysis and by decreased transcript levels. Importantly, the expression of dCas9 alone (i.e. without a gRNA) had minimal impact on chlamydial growth or development. We also describe complementation of the knockdown effect by introducing a transcriptional fusion of the target gene 3’ to the dCas9. Finally, we demonstrate the functionality of a second CRISPRi system based on a dCas12 system that expands the number of potential chromosomal targets. These tools should provide the ability to study essential gene function in Chlamydia.