Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in the developmental cycle.

is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes to developmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal transcription in without detected toxicity of dCas12. Repression of impaired the growth of mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of was downregulated while early genes maintained their expression. Importantly, the growth defect associated with knockdown was rescued by overexpressing at an appropriate degree and time, directly linking the growth patterns to the levels of expression. Interestingly, knockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. with knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes of .