In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Genetics

Background:

• Mitochondrial DNA (mtDNA) mutations are implicated in numerous human diseases.
• Accurate in vivo visualization of cells with mtDNA mutations is crucial but challenging for disease research.

Purpose of the Study:

• To develop a novel imaging system for efficient detection and visualization of mtDNA mutations in live biological systems.
• To demonstrate the utility of this system for identifying disease-related genetic variations.

Main Methods:

• Development of an integrated nano Cas12a sensor (InCasor) for mitochondrial delivery.
• Co-delivery of Cas12a/crRNA, reporters, and Mg2+ into mitochondria for targeted mtDNA cleavage.
• Utilizing Cas12a's trans-cleavage activity to generate fluorescent signals from reporter molecules upon detection of single-nucleotide variations (SNVs).

Main Results:

• InCasor successfully visualized mtDNA mutations in live cells and tumor-bearing mouse models.
• Engineered crRNA enhanced Cas12a sensitivity, enabling precise identification of SNVs.
• The system allowed for in vivo identification of tumor tissues and metastases by detecting mutant mtDNAs.

Conclusions:

• InCasor provides an efficient method for sensing and reporting mtDNA SNVs in vivo.
• This CRISPR imaging nanoprobe offers potential for basic research, diagnostics, and gene therapy related to mtDNA mutations.