In Silico Design of gRNA for CRISPR System for Detection of Multidrug Resistant Tuberculosis Using Indian Mycobacterium tuberculosis Genomes: A Computational Study
Abstract
Background Multidrug-resistant tuberculosis (MDR-TB) continues to pose a major challenge to TB elimination in India, where drug resistance and delayed diagnosis contribute significantly to ongoing transmission. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics have emerged as versatile tools, compared to GeneXpert, capable of detecting resistance-associated mutations with rapid turnaround and high accuracy. This study aimed to design and in silico validate Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based guide RNAs (gRNAs) targeting major drug-resistance mutations in Indian Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods Whole-genome mutation profiles were analyzed using TBProfiler, and gRNAs were designed using CHOPCHOP. Off-target evaluation was performed using Cas-OFFinder and Basic Local Alignment Search Tool (BLAST). High-confidence mutations in gyrA, rpoB, katG, rpsL, embB, and ethA were selected based on prevalence in Indian isolates and WHO-defined resistance markers. Results Numerous drug resistance-associated mutations were identified in the drug-resistant tuberculosis genome isolates. The study identified six key genetic mutations identified in MTB isolates that are associated with phenotypic drug resistance, including gyrA (Asp94Gly), rpoB (Ser450Leu), and katG (Ser315Thr). For each of the six genes, the chromosome position, locus ID, mutation type, and…
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