CRISPRi and beyond: studying essential gene function in the obligate intracellular bacterium Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular bacterium that is the leading cause of bacterial sexually transmitted infections (STIs) and preventable infectious blindness. Its unique biphasic developmental cycle comprises an infectious but non-dividing elementary body and a replicative but non-infectious reticulate body. C. trachomatis possesses a reduced genome where more than half of the open reading frames (ORFs) are predicted to code for essential genes, abrogation of which with traditional chromosomal disruption methods is expected to block bacterial growth and developmental cycle progression. However, understanding the function of such genes is critical to expand our knowledge of chlamydial biology and reveal new therapeutic targets. This review aims to compare and contrast four systems developed in the past 5 years for studying essential genes in Chlamydia. These include systems to conditionally knock down or knockout a target gene product using CRISPR interference (CRISPRi), inducible small RNAs (sRNA), fluorescence-reported allelic exchange mutagenesis (FRAEM) with inducible complementation of the target gene, and dependence on plasmid expression (DOPE).