Conditional dCas12-Mediated Gene Knockdown and Complementation in Chlamydia.

We describe an oligonucleotide RNA-guided, DNase-dead CRISPR-associated protein 12 (dCas12)-mediated transcriptional interference (CRISPRi) system optimized for gene knockdown in Chlamydia trachomatis. In this system, while guide RNAs are expressed constitutively, dCas12 expression is conditional and depends on the presence of the small molecule anhydrotetracycline (ATC). This design enables temporal control of gene repression, allowing the functional analysis of a wide range of genes, including essential genes, without permanent genetic disruption. Due to its T-rich protospacer adjacent motif (PAM) requirement, dCas12 is particularly well-suited for targeting genes in the highly A/T-rich genomes of pathogenic chlamydiae. We further detail strategies for genetic complementation through co-expression of dCas12-resistant alleles, thereby permitting validation of knockdown phenotypes.