Droplet-digital Cas13a assay enables direct single-molecule microRNA quantification

The direct quantification of microRNA at the single-molecule level is still challenging. Herein, we developed a droplet-digital Cas13a assay (ddCA) as a general approach for the amplification-free and absolute quantification of single unlabeled miRNA molecules with single-nucleotide specificity. We demonstrate its simplicity, precise quantification capability, excellent specificity and broad applicability by analyzing microRNAs from synthetic and cell lines-derived materials.