Molecular Docking and Molecular Dynamic Simulation of New Single Guide RNA From Tail Fiber Gene (Gp019) Bacteriophage Salmonella

In this study, we performed in-silico single guide RNA (sgRNA) construction as the first step of the genome editing process using CRISPR-Cas9 on Salmonella SSE-121 phage. The target gene used is a tail fiber protein that plays a role in recognizing and attaching to host bacteria. By carrying out in-silico sgRNA construction, it is expected to be able to determine the optimal sgRNA candidate in Salmonella phage and minimize failure. The genome sequence of Salmonella phage was taken from NCBI and Cas9 protein data was taken from RCSB Protein Data Bank (PDB). The results of sgRNA prediction from Salmonella phage using CHOPCHOP obtained 439 data. Based on the efficiency score, GC content and self complementarity for each candidate, 58 selected sgRNA data were obtained. Selected candidates were selected based on docking score using HNADOCK website and 33 selected candidates were re-docked with Cas9 protein using HDOCK website. The five best candidates were then validated to obtain the most optimal sgRNA candidate. Plasmid construction was performed by matching the plasmid structure with the candidate sgRNA to be inserted and used in in-vivo studies. Based on the data obtained, the docking score for sgRNA and target DNA binding was -553.09, while the docking score for sgRNA and Cas9 binding was -399.18. The plasmid construct pCMV-T7-ABE8 can be well bound by sgRNA 15 using the restriction enzyme SnaBI.