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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.

March 2021Ethics oversight 2020(100)).Note that full information on the approval of the study protocol must also be provided in the manuscript.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA (lncRNA) located in the cell nucleus, is a critical regulator of tumor cell migration. Antisense oligonucleotides (ASOs), which can downregulate the expression level of specific RNAs, have been used in clinical for disease treatment. Herein, we constructed MALAT1-specific ASO and nucleus-targeting TAT peptide cofunctionalized Au nanoparticles, namely, ASO−Au− TAT NPs, which stabilized the fragile ASOs, enhanced nuclear internalization, and exhibited good biocompatibility. After treatment with the ASO−Au−TAT NPs, A549 lung cancer cells showed a greatly reduced MALAT1 expression level and decreased migration ability in vitro. Moreover, the ASO−Au−TAT NPs significantly reduced metastatic tumor nodule formation in vivo. Our results demonstrate that the ASO−Au−TAT nanostructures (NSs) have great potential for treatment of cancer metastasis.