Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrhoeal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by sgRNA to introduce targeted C➔T transitions, resulting in internal STOP codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter assay, we were able to generate loss-of-function mutants in both E. coli and Shigella with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion is in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Further, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella genes and found the mutant phenotype to be similar to that of the reported gene deletion mutants, with no apparent polar effects. This method can be used in a 96-well plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days.ImportanceLoss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches system biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen – Shigella. This pathogen causes high morbidity and mortality in children and antibiotic resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method can be programmed to generated multiple mutants in a single, massively parallel, reaction. By the virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches.