Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India.
Department of Urology, University of Alabama at Birmingham, Hugh Kaul Genetics Building, Birmingham, AL, USA.
Sci Rep. 2021 Sep 7;11(1):17801. doi: 10.1038/s41598-021-97224-4.
Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.
尿路感染（UTI）是世界上最常见的传染病。由于抗生素耐药性的出现，治疗变得越来越困难。因此，迫切需要开发新型抗毒治疗方法来对抗多药耐药的病原菌。规律成簇间隔短回文重复序列（CRISPR）的发现彻底改变了靶向基因编辑技术。CRISPR/Cas9 的最大障碍是货物输送系统，病毒和质粒方法都有缺点。在这里，我们报告了一种基于 CRISPR 的高效新型基因编辑策略 CRISPR-dots，用于靶向毒力因子菌毛粘附（papG 基因），即细菌粘附分子。碳量子点（CQD）被用作 Cas9 和靶向 papG 的向导 RNA（gRNA）进入 UPEC 菌株 CFT073 的输送载体。CQD 通过 DLS 和透射电子显微镜被共价偶联到 Cas9 和靶向 papG 的 gRNA 上，形成纳米复合物 CRISPR-dots（Cri-dots）。Cri-dots-papG 显著靶向 papG，表现为 papG 表达下降。进一步研究表明，papG 缺失的 UPEC 粘附能力和生物膜形成能力显著降低，这通过荧光显微镜和扫描电子显微镜证实。此外，papG 缺失的 UPEC 的毒力降低，表现为秀丽隐杆线虫（C. elegans）存活显著增加与 UPEC 相比。我们的研究结果表明，使用 Cri-dots 纳米复合物靶向 papG 基因显著降低了 UPEC 的致病性。因此，Cri-dots 纳米复合物为对抗多药耐药的 UPEC 提供了一种新型的抗菌策略。
