School of Pharmaceutical Sciences, Collaborative Innovation Center of New Drug Research and Safety Evaluation, Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Zhengzhou University, Zhengzhou 450001, China.
Department of Pathogen Biology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China.
Anal Chem. 2022 Aug 30;94(34):11745-11752. doi: 10.1021/acs.analchem.2c01208. Epub 2022 Aug 17.
Direct visualization of single-nucleotide variation (SNV) in single cells is of great importance for understanding the spatial organization of genomes and their relationship with cell phenotypes. Herein, we developed a new strategy for visualizing SNVs in a nuclear genome using colocalization of dual-engineered CRISPR probes (CoDEC). By engineering the structure of sgRNA, we incorporated a hairpin in the spacer domain for improving SNV recognition specificity and a loop in the nonfunctional domain for localized signal amplification. Using guide probe-based colocalization strategy, we can successfully distinguish on-target true positive signals from the off-target false positives with high accuracy. Comparing with a proximity ligation-based assay (CasPLA), the probe colocalization strategy extended applicable target gene sites (the distance between two designed probes can be extended to around 200nt) and improved detection efficiency. This newly developed method provides a facile way for studying in situ information on SNVs in individual cells for basic research and clinical applications with single-molecule and single-nucleotide resolutions.
直接可视化单细胞中单核苷酸变异（SNV）对于理解基因组的空间组织及其与细胞表型的关系具有重要意义。在此，我们开发了一种使用双工程 CRISPR 探针共定位（CoDEC）在核基因组中可视化 SNV 的新策略。通过工程化 sgRNA 的结构，我们在间隔区中引入发夹结构以提高 SNV 识别特异性，并在非功能区中引入环以实现局部信号放大。通过基于向导探针的共定位策略，我们可以以高精度从脱靶假阳性中成功区分靶标真阳性信号。与基于邻近连接的测定法（CasPLA）相比，探针共定位策略扩展了适用的靶基因位点（两个设计探针之间的距离可延长至约 200nt）并提高了检测效率。该新开发的方法为单细胞中单核苷酸分辨率的基础研究和临床应用中 SNV 的原位信息提供了一种简便的研究方法。
