CRISPR-Cas Single-Guide RNA Construction for Tail Fiber Gene Gp0047 of Salmonella Phage SSE-121

In this study, we conducted an in-silico study of single guide RNA (sgRNA) construction as the initial stage of CRISPR-Cas9-based genome editing on Salmonella bacteriophage SSE-121 to broaden its host range. The results of the study discovered 188 sgRNA candidates using CHOPCHOP prediction. All selected candidates were docked using free website-based docking tools HNADOCK, therefore the top seven candidates of sgRNAs were docked using HDOCK with the Cas9 protein. The molecular dynamics simulation of the most optimum sgRNA-Cas9 protein (docking score -387.43) and sgRNA-Cas9-DNA (docking score -431.58) were calculated using AMBER14 forcefield in YASARA Dynamics. Based on the in-silico evaluation, the gRNA6 was obtained as the optimum single guide RNA. The docking score with DNA targets was -592.23 and Cas9 protein was -387.43. The comparison between the Cas9 and Cas9-sgRNA complex showed that the binding effect of sgRNA could maintain better conformational stability than in the single form.