School of Pharmaceutical Sciences, Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Collaborative Innovation Center of New Drug Research and Safety Evaluation, State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou University, Zhengzhou, 450001, China.
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, 100084, China.
Biosens Bioelectron. 2022 Nov 15;216:114641. doi: 10.1016/j.bios.2022.114641. Epub 2022 Aug 19.
Bacterial genotyping is important for understanding the complex microbiota. Although fluorescence in situ hybridization (FISH) has enabled bacterial community identification with high spatial resolution, its unavoidable cell fixation steps and signal generation by multi-probe stacking greatly limit its application in living bacterial genotyping. Here, we designed polyethyleneimine-encapsulated CRISPR/Cas12a-circular reporter nanoprobes (CasCLR) for rapid and sensitive visualization of gene information in living bacteria. We found that, nanoprobe-based sequential delivery of Cas12a/crRNA and circular reporter into bacteria allowed single genomic loci to initiate trans-cleavage activity of Cas12a, thereby cleaving CLR to generate amplified fluorescent signals for imaging of target gene. Using CasCLR, we can sensitively analyze the percentage of target bacteria in co-culture experiments and directly detect pathogenic bacteria in uncultured mouse gut microbe. In addition, CasCLR has the ability to sensitively analyze specific genotype of microbial communities in vivo. This nanobiotechnology-based bacterial gene analysis is expected to advance understanding of in vivo bacterial cytogenetic information.
细菌基因分型对于了解复杂的微生物群落非常重要。虽然荧光原位杂交（FISH）能够以高空间分辨率识别细菌群落，但它不可避免的细胞固定步骤和多探针堆积产生的信号极大地限制了其在活细菌基因分型中的应用。在这里，我们设计了聚乙烯亚胺包裹的 CRISPR/Cas12a-环形报告子纳米探针（CasCLR），用于快速灵敏地可视化活细菌中的基因信息。我们发现，基于纳米探针的 Cas12a/crRNA 和环形报告子的顺序递送至细菌中，允许单个基因组位点启动 Cas12a 的跨切割活性，从而切割 CLR 以产生扩增的荧光信号，用于目标基因的成像。使用 CasCLR，我们可以在共培养实验中灵敏地分析目标细菌的百分比，并直接检测未培养的小鼠肠道微生物中的致病菌。此外，CasCLR 还具有在体内灵敏分析微生物群落特定基因型的能力。这种基于纳米生物技术的细菌基因分析有望促进对体内细菌细胞遗传学信息的理解。
