Direct Visualization of Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation Assay

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.

A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

“…This unique conformational rearrangement may provide an ideal targeting site for various isothermal amplification techniques with enhanced robustness, specificity, and sensitivity due to the intrinsic properties of CRISPR effectors. For example, when combined with the exponential amplification reaction (EXPAR) and rolling cycle amplification (RCA), the CRISPR effector, Cas9, has been successfully applied in the efficient pre-screening of sgRNAs 27 and sensitive in situ genomic loci detection 28 , respectively. Thus, this CRISPR effectors-triggered strategy has great potentials to be applied in different situations.…”

Visualizing, quantifying, and manipulating mitochondrial DNA in vivo

“…In this technique, Cas9/gRNA is directed to a specific sequence on mtDNA and local amplification of a fluorescence signal is achieved via a proximity ligation assay. This can enable visualization of individual nucleoids containing the specific mtDNA sequence of interest (63). The low copy number of mtDNAs in nucleoids (25) confirms that CasPLA can achieve high sensitivities at the level of single molecules.…”

“…Labelling of mtDNA containing single-nucleotide variations has also been achieved in situ using a technique termed CasPLA (63). In this technique, Cas9/gRNA is directed to a specific sequence on mtDNA and local amplification of a fluorescence signal is achieved via a proximity ligation assay.…”

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

“…For example, some newly developed CRISPR systems (such as Cas12a and Cas13a) have now be used for genotypic and phenotypic analysis of bacterial pathogens. 47,[65][66][67][68][69][70] Specically, a CRISPR-Cas13a/C2c2-based SHERLOCK platform has been applied for ultrafast and sensitive RNA/DNA quantitation, which is suitable for rapid identication of bacterial pathogens by detection of specic bacterial genes. 66 Recently, an allosteric probe-initiated catalysis and CRISPR-Cas13a amplication reaction (termed 'APC-Cas') was developed for detecting very low numbers of a bacterial pathogens without isolation 71 and can selectively quantify bacterial cells with a concentration from 1 to 10 5 CFU mL À1 in various types of samples, and the cost of reagents can be reduced to below $1 per test.…”