CRISPR-CAS-BASED FUNCTIONAL GENOMICS FOR IDENTIFICATION OF GENETIC DETERMINANTS GOVERNING ANTIMICROBIAL RESISTANCE IN MULTIDRUG-RESISTANT BACTERIA

Abstract

The growing threat of antimicrobial resistance (AMR), particularly among multidrug-resistant bacteria, underscores the urgent need for innovative approaches to identify genetic vulnerabilities and potential therapeutic targets. Traditional methods, such as transposon insertion sequencing (Tn-seq), have significantly advanced our understanding of bacterial gene essentiality and the genetic determinants required for survival. They can't look at genes that are essential because changing them would kill the bacteria. CRISPR and CRISPR-associated (Cas) tools have changed the way scientists study genes by making it easier to control gene activity. CRISPR interference (CRISPRi) uses a version of Cas that doesn't cut DNA, called dCas to stop genes from working. This method works for both non-essential genes without breaking DNA. This review looks closely at CRISPR-Cas tools that help find out which genes are important for resistance. It explains how the active Cas systems work compared to the dCas ones and how CRISPRi and CRISPR activation (CRISPRa) can be used to test what happens when genes are turned off or on. The review also covers high-throughput methods for testing genes at once finding genes that work together in a bad way and looking at how genes interact with chemicals to find weak points in dangerous bacteria like Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Mycobacterium tuberculosis. New techniques like using CRISPR tools at once testing all the genes on a plasmid and delivering CRISPR inside bacteria using modified viruses or other delivery systems are also discussed. The study ends with challenges like effects problems with gene groups, issues with how genes are turned on resistance that develops over time and the bodys immune response. Combining CRISPR tools, with research and better delivery systems could help find new ways to fight drug-resistant bacteria.