The CRISPR/Cas9-Mediated Knockout of in Wild Pathogenic to Alleviate the Effects on Cell Damage and Autophagy.

CRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type remains limited. Pathogenic wild-type , a major cause of foodborne illnesses and intestinal inflammation in humans and animals, poses a significant global public health threat. The valine-glycine repeat protein G (VgrG) is a key virulence factor that enhances pathogenicity. In this study, PCR was used to identify 50 strains carrying the virulence gene out of 83 wild pathogenic strains, with only one strain sensitive to kanamycin and spectinomycin. A homologous repair template for was constructed using overlap PCR. A dual-plasmid CRISPR/Cas9 system, combining pTarget (spectinomycin resistance) and pCas (kanamycin resistance) with Red homologous recombination, was then used to induce genomic cleavage and knock out . PCR and sequencing confirmed the deletion of a 1708 bp fragment of the gene in wild-type . IPEC-J2 cells were infected with -WT and ∆, and treated with the mTOR inhibitor rapamycin to study the effects of on the mTOR signaling pathway. The qPCR results showed that activated the mTOR pathway, suppressed and mRNA levels, and upregulated the autophagy-related genes and LC3-II protein expression. In conclusion, we utilized CRISPR/Cas9 technology to achieve large-fragment deletions in wild-type , revealing that activates the mTOR signaling pathway and upregulates autophagy markers. These findings offer new insights into genome editing and clarifies the pathogenic mechanisms through which induces cellular damage.