Concurrent Detection\nof Protein and miRNA at the\nSingle Extracellular\nVesicle Level Using a Digital Dual CRISPR-Cas Assay

The simultaneous detection of proteins and microRNA (miRNA)\nat\nthe single extracellular vesicle (EV) level shows great promise for\nprecise disease profiling, owing to the heterogeneity and scarcity\nof tumor-derived EVs. However, a highly reliable method for multiple-target\nanalysis of single EVs remains to be developed. In this study, a <b>d</b>igital <b>d</b>ual CRISPR-Cas-powered <b>S</b>ingle <b>E</b>V <b>E</b>valuation (<b>ddSEE</b>) system was proposed to enable the concurrent detection of surface\nprotein and inner miRNA of EVs at the single-molecule level. By optimizing\nsimultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a,\nthe surface protein of EVs was detected by Cas12a using antibody-DNA\nconjugates to transfer the signal of the protein to DNA, while the\ninner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic\nchip containing 188,000 microwells was used to convert the CRISPR-Cas\nsystem into a digital assay format to enable the absolute quantification\nof miRNA/protein-positive EVs without bias through fluorescence imaging,\nwhich can detect as few as 214 EVs/μL. Finally, a total of 31\nblood samples, 21 from breast cancer patients and 10 from healthy\ndonors, were collected and tested, achieving a diagnostic accuracy\nof 92% in distinguishing patients with breast cancer from healthy\ndonors. With its absolute quantification, ease of use, and multiplexed\ndetection capability, the ddSEE system demonstrates its great potential\nfor both EV research and clinical applications.