In silico design of sgRNAs with knockout potential for the adeB gene of the AdeABC efflux pump of Acinetobacter baumannii.

Abstract

Background: The efflux pump AdeABC, regulated by the AdeRS operon, is a key factor in multidrug resistance in Acinetobacter baumannii. The adeB gene encodes the transmembrane transporter protein, making it a promising target for therapeutic gene disruption.

Methods: We designed and analyzed single guide RNAs (sgRNAs) targeting the adeB gene using CHOPCHOP. Conservation analysis was conducted with BLAST and multiple alignment tools. Potential off-target effects were assessed using Cas-OFFinder. The thermodynamic stability and accessibility of top-ranked sgRNAs were evaluated using Mfold. Structural and functional domain mapping was performed based on the 3D model of AdeB protein. Phylogenetic analyses of adeB alleles were carried out using MAFFT and iTOL.

Results: Among 49 designed sgRNAs, five candidates met stringent criteria for cleavage efficiency, GC content, conservation across strains, and low off-target potential. Secondary structure analysis confirmed their stability, and domain mapping showed that the sgRNAs targeted critical regions of AdeB, including transmembrane helices and substrate-binding sites. Phylogenetic clustering confirmed high sequence conservation in clinical strains.

Conclusion: This study presents three validated sgRNAs with knockout potential against the adeB gene of A. baumannii. These sgRNAs may serve as effective molecular tools to disrupt the AdeABC pump and combat efflux-mediated antimicrobial resistance. Further in vitro validation is proposed to assess their functional impact.