Objectives: Antibiotic resistance in Helicobacter pylori ( H. pylori ) has reduced the effectiveness of standard eradication regimens, highlighting the need for alternative antimicrobial strategies. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) offers sequence-specific genome targeting, but rational application requires systematic computational target prioritization. Methods: We developed a reproducible bioinformatics framework to prioritize CRISPR-Cas9 guide Ribonucleic acids (RNAs) (gRNAs) targeting conserved core fitness genes ( recA, ureA ) and virulence- or colonization-associated genes ( cagA, vacA, flgE ) in H. pylori . Guide design, thermodynamic stability assessment, off-target screening, gene conservation analysis, and pathway/network association analyses were performed. Results: Twelve high-confidence gRNAs met predefined efficiency and specificity criteria. Core fitness genes exhibited higher sequence conservation than virulence-associated genes. Pathway and interaction network analyses provided a qualitative context for comparative target prioritization. Conclusion: This study presents a computational framework for prioritizing CRISPR-Cas9 targets in H. pylori . The findings are hypothesis-generating and intended to guide future experimental validation rather than demonstrate therapeutic feasibility.
Shafee Ur Rehman (Sat,) studied this question.