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

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. 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. 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 900-fold compared with the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains.