CRISPR-Based Functional Genomics Screen Identifies Novel Antimicrobial Resistance Determinants in Nigerian Multidrug-Resistant Klebsiella pneumoniae Isolates

Multidrug-resistant Klebsiella pneumoniae harbors a large reservoir of uncharacterized genes that may contribute to resistance phenotypes beyond those encoded by recognized resistance determinants. Functional genomics screens using CRISPR interference (CRISPRi) offer a powerful approach to systematically identify novel genes contributing to antibiotic resistance, generating new targets for drug development and diagnostic innovation. This study designed and implemented a CRISPRi-based genome-wide fitness screen in MDR K. pneumoniae isolates obtained from tertiary hospitals in Kano, Abuja, and Enugu to identify novel determinants of resistance to carbapenems, colistin, and tigecycline. A CRISPRi library of guide RNAs targeting 4,200 K. pneumoniae genes was constructed and introduced into five representative MDR clinical isolates. Pooled screens under antibiotic selective pressure were followed by next-generation sequencing to identify depleted guide RNAs corresponding to resistance-conferring genes. Candidate novel determinants were validated by individual gene knockdown and overexpression experiments. The screen identified 23 candidate novel resistance determinants not previously reported in K. pneumoniae. Thirteen of these were validated as contributing to clinically meaningful resistance phenotype changes upon knockdown. Of particular interest, a previously uncharacterized outer membrane protein (designated OmpK-R1) contributed to carbapenem impermeability through porin channel occlusion, confirmed by electrophysiology experiments. These findings represent an original functional genomics contribution to K. pneumoniae resistance biology and generate novel candidate targets for the development of resistance-breaking therapeutic strategies applicable to Nigerian clinical isolates. Keywords: CRISPRi, Klebsiella pneumoniae, functional genomics, antimicrobial resistance, novel determinants.