Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.

Automated summary

This study presents a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level, achieving high sensitivity and specificity through optimized reaction parameters and microdroplet technology. The method can directly count diverse viruses' DNAs and demonstrate versatility and quantification capability, showing potential as a versatile and quantitative platform for molecular diagnosis.