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Direct Visualization of Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation Assay.
- Source :
-
Journal of the American Chemical Society [J Am Chem Soc] 2018 Sep 12; Vol. 140 (36), pp. 11293-11301. Date of Electronic Publication: 2018 Aug 30. - Publication Year :
- 2018
-
Abstract
- 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.
Details
- Language :
- English
- ISSN :
- 1520-5126
- Volume :
- 140
- Issue :
- 36
- Database :
- MEDLINE
- Journal :
- Journal of the American Chemical Society
- Publication Type :
- Academic Journal
- Accession number :
- 30125486
- Full Text :
- https://doi.org/10.1021/jacs.8b05309