Harnessing the Native Type I-F CRISPR-Cas System of Acinetobacter baumannii for Genome Editing and Gene Repression

Introduction: The rapid emergence of infections caused by multidrug-resistant Acinetobacter baumannii ( A. baumannii ) has posed a serious threat to global public health. It has therefore become important to obtain a deeper understanding of the mechanisms of multidrug resistance and pathogenesis of A. baumannii ; however, there are still relatively few genetic engineering tools for this. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. Methods: A single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii was developed. The protospacer adjacent motif sequence was identified as 5 ' -NCC-3 ' by analysis of the CRISPR array. Results: Through introduction of the RecAb homologous recombination system, the knockout efficiency of the oxyR gene significantly increased from 12.5% to 75.0% in A. baumannii . To investigate transcriptional inhibition by the Type I-F CRISPR system, the gene encoding its Cas2-3 nuclease was deleted and the native Type I-F Cascade effector was repurposed to regulate transcription of alcohol dehydrogenase gene adh4 . The level of adh4 transcription was inhibited by up to 90 0-fold compared with the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. Conclusion: This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains. (c) 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.