Patients with pathogenic mitochondrial DNA (mtDNA) point mutations often exhibit debilitating neurological syndromes, leading to severe disability and premature death. At least 1 in 5000 individuals are affected by mtDNA diseases. Mitochondrial genetic diseases exhibit heteroplasmic single nucleotide polymorphisms (SNPs), where wild-type and mutant types coexist. The heteroplasmy rate varies among individuals, affecting clinical symptoms. Homoplasmic mtDNA mutations are easily detected in blood, but heteroplasmic mutations vary in percentage across tissues and over time. Accurate detection of SNP heteroplasmy levels is crucial for diagnosing mitochondrial genetic diseases. CRISPR-based molecular diagnostics have gained attention for their specificity, sensitivity, and speed. However, single signal output methods struggle to accurately diagnose the heteroplasmy levels of mtDNA diseases due to template quantity in samples. Precise SNP abundance quantification remains a challenge.

This research by Associate Professor Liu Yizhen's team at the School of Chemical and Environmental Engineering, published in "Biosensors and Bioelectronics," presents RatioCRISPR, an automated CRISPR/Cas12a biochip sensor. It quantifies heteroplasmic SNP levels in mitochondrial diseases. DNA is extracted from patient blood samples and mixed with RPA amplification reagents, then added to the chip. The chip runs automatically, using a ratiometric signal output to minimize sample concentration effects, focusing on the relative amounts of wild-type and mutant templates. RatioCRISPR can detect 8 samples in 25 minutes with a limit of detection (LOD) of 15.7 aM. It successfully detected 13 simulated samples of 3 mtDNA point mutations, setting a heteroplasmy threshold (60%) for disease risk assessment. This automated, precise biosensor is applicable for diagnosing multiple SNPs, especially heteroplasmic ones, offering a fast, economical, real-time clinical diagnosis platform for genetic counseling, epidemiological studies, and various heteroplasmic SNP diseases.

Link: https://doi.org/10.1016/j.bios.2023.115676

The study introduces RatioCRISPR, an automated CRISPR/Cas12a ratiometric biochip sensor, for precise quantification of heteroplasmic SNPs in mitochondrial diseases. The process involves extracting DNA from mtDNA disease patient blood samples, mixing with RPA amplification reagents, and adding to the chip, which runs automatically. RatioCRISPR employs a ratiometric signal output for stability and minimal sample concentration impact, focusing on relative amounts of wild-type and mutant templates. It accurately detects 8 samples within 25 minutes with a detection limit of 15.7 aM, successfully identifying 13 simulated samples of 3 mtDNA point mutations, crucial in assessing disease risks. This automated, precise biosensor offers a rapid, cost-effective, real-time clinical diagnosis platform for genetic counseling, epidemiological studies, and various heteroplasmic SNP diseases.

Figure 1: Schematic Diagram of RatioCRISPR for mtDNA Disease Diagnosis

The authors propose a universal, automated RPA-CRISPR/Cas12a ratiometric assay chip, RatioCRISPR, for diagnosing mitochondrial DNA diseases. This approach, introduced for the first time in CRISPR/Cas-based biochips, achieves precise quantification of SNP relative abundance. Advantages of this platform include: 1) Ratio-based signal output system for quantifying heteroplasmy levels, reducing the impact of sample concentration; 2) An integrated, closed chip design, preventing aerosol contamination and enabling multiplexed automated detection. This advanced, convenient biosensor is versatile, highly automated, and accurate, showing great practicality and market potential for diagnosing multiple SNPs, particularly those with heteroplasmic variations.