CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis

Abstract

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.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

“…An alternative hypothesis that may explain our findings is offered by recent evidence obtained by Ouellette et al (2021) who found that introducing a weak ribosome binding site overcomes leaky protein expression from the tet promoter 63 . Further testing and optimisation of riboswitches in Chlamydia is needed to resolve this, and alternative riboswitch designs (and perhaps a Chlamydia -specific riboswitch) may be required if they are to offer an alternative to the tet promoter for inducible protein expression in Chlamydia .…”

The Chlamydia trachomatis inclusion membrane protein CT006 associates with lipid droplets in eukaryotic cells

“…The latter is supported by the fact that a previous large mutagenesis screen did not isolate nonsense mutations for ct006 [ 24 ]. The recent development of a CRISPR interference system enabling induced and transient repression of chlamydial genes [ 51 ] should help clarifying this issue but this is beyond the scope of this manuscript.…”

Expression and structure of the Chlamydia trachomatis DksA ortholog

“… 2018 , Keb and Fields 2020 ) or CRISPR interference (Ouellette 2018 , Ouellette et al . 2021 ) in case dksA Ct is essential to C. trachomatis intracellular replication and/or developmental transitions. Mechanistic understanding of how DksA Ct functions in C. trachomatis biology will require analysis of whether the protein directly influences pathogen gene expression by binding to RNAP akin to prototypical DksA proteins, or whether DksA Ct is associated with a different mechanism of action, such as interfering with the binding of another protein to RNAP, thus indirectly affecting transcriptional regulation.…”