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