CRISPR interference of essential stage-specific gene function in Chlamydia trachomatis

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CRISPR interference of essential stage-specific gene function in Chlamydia trachomatis Abstract Chlamydia trachomatis is a clinically important obligate intracellular bacterial pathogen and the leading cause of preventable infectious blindness (trachoma) and bacterial sexually transmitted diseases worldwide. If not cleared, then chlamydial infections can lead to poor health consequences, such as pelvic inflammatory disease and tubal factor infertility in women. Despite its devastating impacts on human health, much of C. trachomatis pathobiology remains unknown. Whereas the characteristics of the developmental cycle of C. trachomatis are well studied, the regulatory pathways and mechanisms remain poorly defined, undermining fundamental understanding of C. trachomatis pathogenesis. It is extremely challenging to study essential genes, whose disruption leads to lethality. To address this gap, we have successfully adapted CRISPR interference (CRISPRi) gene silencing technology, which relies on inducible expression of catalytically inactive variants of dCas orthologs that repress gene transcription at a specific chromosomal locus in C. trachomatis. This approach allows for creation of conditional, reversible knockdowns in individual genes that can also be complemented by including a plasmid-encoded copy of the targeted gene as a transcriptional fusion with the dCas ortholog. Building on these findings, we have further adapted our CRISPRi approaches to allow for multiplexed gene knockdown for the targeted knockdown of multiple genes simultaneously. The objective of the proposed study is to develop a next generation of CRISPR interference (CRISPRi) genetic editing toolbox in C. trachomatis and to explore its utility towards elucidating the underlying mechanisms of developmental gene regulation. In Specific Aim 1, we will establish an adaptable second generation of CRISPRi platform, which enables tunable, trackable, and quantitative assessment of CRISPRi-mediated changes in C. trachomatis cellular and molecular phenotypes in single cells. In Specific Aim 2 we will test our hypothesis that a set of “known” and “unknown” genes are essential for critical events of ending replication of reticulate bodies (RB) and RB differentiating to the infectious elementary body (EB). Leveraging CRISPRi, this Aim will elucidate the relative contributions of each essential gene product to C. trachomatis developmental processes critical for EB formation and reinitiation of a new cycle of infection. Collectively, this innovative research project will seek a focused investigation to illustrate what chlamydial factors are crucial for, and how these factors directly contribute to, regulation of the late developmental cycle of C. trachomatis. It is also significant, as the methods developed will be the starting point towards interrogation of genome-wide studies of gene function and genetic interactions in C. trachomatis.