Droplet Digital CRISPR for Nucleic Acid Detection

Abstract

Droplet digital (dd) clustered regularly interspaced short palindromic repeats (CRISPR) integrates the high sequence specificity of CRISPR-based nucleic acid detection with the absolute quantification capability of digital droplet microfluidics, offering high sensitivity, precision, and scalability. By partitioning samples into thousands to millions of picoliter microdroplets, ddCRISPR enables single-molecule resolution and minimizes background interference. This review summarizes the principles of droplet generation, manipulation, and detection in ddCRISPR platforms, as well as recent advances in amplification-based and amplification-free detection strategies. Representative applications are highlighted for viral, bacterial, and other DNA/RNA biomarker detection. Current challenges, including workflow automation, droplet stability, multiplexing, and assay portability, are discussed alongside future perspectives such as artificial intelligence (AI)-assisted analysis, point-of-care integration, and high-throughput multiplexed detection. These insights aim to guide the translation of ddCRISPR technologies from laboratory research to robust, scalable, and accessible diagnostic solutions.

Keywords: CRISPR/Cas detection; absolute quantification; droplet digital microfluidics; nucleic acid biomarkers.