In Silico Study of CRISPR-Cas Single-Guide RNA Construction for Salmonella Phage

Abstract

The majority of cases of foodborne disease are caused by Salmonella. Bacteriophage therapy is a solution to overcome Salmonella infection without induced antibiotic resistance effects. To enhance the host range of bacteriophage infection is using genome editing. In this study, we conducted an in-silico study of single guide RNA (sgRNA) construction as the initial stage of genome editing using the CRISPR-Cas9 system on Salmonella bacteriophage SSE-121 to broaden its host range. The target gene in this study was a tail fiber protein that plays a role in the process of recognizing and attaching viruses to the bacterial hosts. The Salmonella phage genome was obtained from the NCBI gene bank (NCBI Reference Sequence: NC_027351.1) and the Cas9 protein from RCSB PDB Protein Data Bank (PDB ID: 4ZT0). The results of the study were 188 sgRNA candidates using CHOPCHOP website (http://chopchop.cbu.uib.no/). Fourteen sgRNA candidates were selected based on efficiency score, GC content, and self-complementarity. Further selection to obtain the optimum sgRNA was carried out based on molecular docking score. All selected candidates were docked using website docking-based tools HNADOCK (http://huanglab.phys.hust.edu.cn/hnadock/) and the top 7 candidate sgRNAs were docked using HDOCK (http://hdock.phys.hust.edu.cn/) with the Cas9 protein. In summary, the in-silico study for sgRNA construction can determine the optimum sgRNA for Salmonella phage, and its expected to minimize failures and errors during in-vivo experiments.