CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in <i>Coxiella burnetii</i>

Understanding the genetic mechanisms that enable <i>C. burnetii</i> to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in <i>C. burnetii</i> using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in <i>C. burnetii</i> that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for <i>C. burnetii</i> host infection and disease.