State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; College of Life Science, University of Chinese Academy of Sciences, Beijing, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Int J Antimicrob Agents. 2023 Nov;62(5):106962. doi: 10.1016/j.ijantimicag.2023.106962. Epub 2023 Sep 4.
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.
多重耐药鲍曼不动杆菌（A.baumannii）引起的感染迅速出现，对全球公共卫生构成了严重威胁。因此，深入了解 A.baumannii 的多药耐药机制和发病机制变得尤为重要；然而，目前用于该菌的遗传工程工具仍然相对较少。尽管 A.baumannii 拥有 I 型-F CRISPR-Cas 系统，但尚未将其用于遗传修饰。
开发了一种用于 A.baumannii 基因编辑和基因调控的单质粒介导的天然 I 型-F CRISPR-Cas 系统。通过对 CRISPR 阵列的分析，确定前间区序列基序序列为 5'-NCC-3'。
通过引入 RecAb 同源重组系统，oxyR 基因的敲除效率从 A.baumannii 中的 12.5%显著提高到 75.0%。为了研究 I 型-F CRISPR 系统的转录抑制作用，删除了编码其 Cas2-3 核酸酶的基因，并重新利用天然 I 型-F Cascade 效应物来调节醇脱氢酶基因 adh4 的转录。adh4 转录水平与对照相比抑制了 900 倍。Cascade 转录模块也成功应用于临床分离的肺炎克雷伯菌。
本研究提出了一种用于未来探索 A.baumannii 或其他临床菌株遗传特征的工具。
