Double-emulsion droplet digital CRISPR/Cas12a for amplification-free, absolute quantification of nucleic acids at attomole levels

• d 3 CRISPR platform enables amplification-free, absolute quantification of nucleic acids. • Stable DE droplets integrate with flow cytometry for high-throughput analysis. • Detects as low as 100 aM HPV DNA and 60 copies/µL E. coli DNA. • Offers a 1,000-fold improvement in LOD over bulk amplification-free Cas12a assays. • Achieves rapid DNA quantification in diverse complex sample matrices. . The quantification of nucleic acids is of prominent importance for biology and medicine sciences. Droplet digital polymerase chain reaction (ddPCR) provides an absolute measure of target nucleic acid molecules with unrivalled sensitivity and accuracy, but suffers from limitations inherent to PCR amplification, droplet partition, and signal detection. Here, we present an ultrasensitive, rapid, and high-throughput technique for the absolute quantification of nucleic acids without the need for amplification, by combining the d ouble-emulsion (DE) d roplet d igital platform with CRISPR /Cas12a system (d 3 CRISPR). We demonstrate the developed approach by accurately quantifying various DNA molecules, such as target human papillomavirus (HPV) 18, HPV16, and E. coli DNA, at concentrations down to attomole levels. This represents an over 1,000-fold improvement in the limit of detection (LOD) compared to existing bulk amplification-free Cas12a assays. Given the versatility and generality of the CRISPR system, we believe that this approach has great potential in the detection and measurements of diverse nucleic acid molecules for many biomedical, clinical, and environmental applications.