Visualizing Single-Nucleotide Variations in a Nuclear Genome Using Colocalization of Dual-Engineered CRISPR Probes
Visualizing Single-Nucleotide Variations in a Nuclear Genome Using Colocalization of Dual-Engineered CRISPR Probes
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使用双工程 CRISPR 探针的共定位可视化核基因组中的单核苷酸变异
DOI：
10.1021/acs.analchem.2c01208
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发表时间：
2022
影响因子：
7.4
通讯作者：
Jinghong Li
中科院分区：
文献类型：
--
作者：
Yan Liang;Sixuan Wu;Wenshuai Han;Jinjin Wang;Chenlu Xu;Jinjin Shi;Zhenzhong Zhang;Hua Gao;Kaixiang Zhang;Jinghong Li
直接显示单个细胞中的单核苷酸变异(SNV)对于了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。在这里，我们开发了一种新的策略，使用双工程CRISPR探针(编解码器)的共定位来可视化核基因组中的SNV。通过改造sgRNA的结构，我们在间隔域加入了一个发夹，以提高SNV识别的特异性，并在非功能域加入了一个环，用于局部信号放大。使用基于引导探针的共定位策略，可以很好地区分目标上的真阳性信号和目标外的假阳性信号，并且准确率很高。与基于邻近连接的分析方法(Caspla)相比，探针共定位策略扩展了适用的靶基因位点(设计的两个探针之间的距离可延长至200nT左右)，并提高了检测效率。这种新开发的方法为单分子和单核苷酸分辨率的基础研究和临床应用提供了一种简单的方法来研究单个细胞中SNV的原位信息。
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.