Moein Iranmanesh - Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
Abbas Hajizade - Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
Mohammad Hashemabadi - Department of Genetic, Faculty of Sciences, Tarbiat Modares University, Tehran, Iran
Yosof Tarverdizadeh - Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
Mostafa Zare - Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
Mohsen yaghoubi - Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran

Shigella sonnei is a common bacterial pathogen responsible for gastrointestinal infections in humans. The development of new therapeutic strategies to combat Shigella sonnei infections is of great importance. In this study, we used the CRISPR-Cas۹ gene editing system to target and delete the MxiE gene in Shigella sonnei. The MxiE gene encodes a transcriptional regulator that plays an important role in the pathogenicity of Shigella sonnei. By disrupting the function of this gene, we aimed to discover its precise contribution to bacterial virulence.To achieve deletion of the MxiE gene, we used the CRISPR-Cas۹ system, which enabled precise and efficient gene editing. We designed a guide RNA (gRNA) specific for the target site in the MxiE gene and introduced it into Shigella sonnei strains expressing Cas۹ endonuclease. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial genome following the cleavage of the bacterial genome by Cas۹ enzyme. After the gene editing process, we confirmed the successful deletion of the MxiE gene using PCR analysis, enzyme digestion and DNA sequencing.The results of PCR, restriction digestion and sequencing showed the successful recombination of S. sonnei. We will next investigate the effect of MxiE gene deletion on the pathogenicity of Shigella sonnei through a series of in vitro and in vivo experiments.We hypothesize that a significant reduction in the ability of MxiE deleted strains to invade and induce cytotoxicity in host cells is observed. In the following we will develop a mouse infection model, mice challenged with MxiE deleted strains.This study not only highlights the successful application of the CRISPR-Cas۹ system for gene deletion in Shigella sonnei, but also provides valuable insights into the role of the MxiE gene in bacterial pathogenicity. The findings of this study contribute to a better understanding of the molecular mechanisms underlying Shigella sonnei infections and provide potential avenues for the development of targeted therapeutic interventions.In conclusion, our study demonstrates the efficiency of the CRISPR-Cas۹ system in deleting the MxiE gene in Shigella sonnei, leading to reduced virulence. This research lays the foundation for further research into the development of innovative treatments and preventive measures against Shigella sonnei infections.

CRISPR-Cas۹ gene editing, Gene deletion, Bacterial pathogenesis, Virulence factors