Dissecting multi-gene virulence phenotypes with base editing requires consideration of target-independent mutagenesis

Bacterial pathogenicity arises from complex genetic interactions that are difficult to characterise through single-gene deletions. CRISPR base editors can generate multiplexed gene knockouts, yet this technology remains unexplored for dissecting bacterial pathogenicity. Here, we developed a base-editing pipeline for multi-gene knockouts while revealing that target-independent editing can contribute to variability in clonal fitness. In the model pathogen Salmonella Typhimurium, we employed curable plasmids containing a cytidine deaminase base editor and a multi-spacer CRISPR array to introduce premature stop codons in up to nine genes encoding SPI-2 T3SS effector proteins. Target bases were efficiently edited, producing a multi-knockout strain that showed reduced virulence in vivo relative to single knockouts. However, whole-genome sequencing revealed off-target cytidine deaminase activity, which affected virulence in vivo in a clone-dependent manner. A statistical power analysis predicted how many edited mutants are needed to confidently measure fitness functions in the face of off-target mutations. Our work shows the potential and current limitations of multiplexed base editing in bacterial pathogens and highlights the need for properly addressing off-target mutations when deploying base editors to interrogate genotype-phenotype relationships.