Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation , Tsinghua University , Beijing 100084 , China.
J Am Chem Soc. 2018 Sep 12;140(36):11293-11301. doi: 10.1021/jacs.8b05309. Epub 2018 Aug 30.
The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.
线粒体 DNA（mtDNA）突变在细胞中的积累与衰老相关疾病密切相关。对 mtDNA 中单核苷酸变异（SNV）的成像对于理解携带致病性变化的 mtDNAs 的异质性至关重要。在此，我们设计了一种 CRISPR/Cas9 介导的邻近连接分析（CasPLA），用于直接可视化单个细胞中 mtDNA 的 ND4 和 ND5 基因。利用 CRISPR/Cas9 的高特异性，CasPLA 可用于以单分子分辨率成像 ND4 基因中的 SNV。使用 CasPLA，我们观察到通过隧道纳米管在不同细胞之间发生 mtDNA 转移过程，这可能解释了 mtDNA 异质性的传播。此外，我们证明 CasPLA 策略可用于核基因组中单拷贝基因组基因座（KRAS 基因）的成像。我们的结果确立了 CasPLA 作为一种在单个细胞中进行 SNV 原位研究的工具，用于基础研究和遗传诊断。
