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

Newson, J. P. M.; Gawlitt, S.; Sundar, S.; Regoes, R. R.; Beisel, C.; Hardt, W.-D.

doi:10.64898/2026.02.11.705302

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bioRxiv · 10.64898/2026.02.11.705302

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

Newson, J. P. M.·Gawlitt, S.·Sundar, S.·Regoes, R. R.·Beisel, C.·Hardt, W.-D.

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Abstract

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.

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Newson, J. P. M., Gawlitt, S., Sundar, S., Regoes, R. R., Beisel, C., Hardt, W.-D.. 2026-02-12. Dissecting multi-gene virulence phenotypes with base editing requires consideration of target-independent mutagenesis. https://doi.org/10.64898/2026.02.11.705302

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