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

Abstract

The rapid emergence of infections caused by MDR Acinetobacter baumannii poses a serious threat to global public health. Therefore, it has 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 A. baumannii. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. Here, we developed a single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii. The protospacer adjacent motif (PAM) sequence was identified as 5'-NCC-3' by analysis of the CRISPR array. Through introduction of the RecAb homologous recombination system, we significantly increased the knockout efficiency of the oxyR gene from 12.5% to 75.0% in A. baumannii. To investigate transcriptional inhibition by the Type I-F CRISPR system, we deleted the gene encoding its Cas2-3 nuclease and repurposed the native Type I-F Cascade effector to regulate transcription of alcohol dehydrogenase gene adh4. The level of adh4 transcription was inhibited by up to 900-fold compared to 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.