Massively multiplexed nucleic acid detection with Cas13

• Published in: Nature (London) Vol. 582; no. 7811; pp. 277 - 282
• Main Authors: Ackerman, Cheri M, Myhrvold, Cameron, Thakku, Sri Gowtham, Freije, Catherine A, Metsky, Hayden C, Yang, David K, Ye, Simon H, Boehm, Chloe K, Kosoko-Thoroddsen, Tinna-Sólveig F, Kehe, Jared, Nguyen, Tien G, Carter, Amber, Kulesa, Anthony, Barnes, John R, Dugan, Vivien G, Hung, Deborah T, Blainey, Paul C, Sabeti, Pardis C
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.06.2020; Nature Publishing Group UK
• Subjects: Acids; Animals; Betacoronavirus - genetics; Betacoronavirus - isolation & purification; Codes; Combinatorial analysis; Coronaviruses; COVID-19; CRISPR; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems - genetics; Design; Diagnostic software; Diagnostic systems; Droplets; Drug resistance; Drug Resistance, Viral - genetics; Epidemics; Gene sequencing; Genome, Viral - genetics; Genomes; Glass substrates; Health surveillance; HIV; HIV - classification; HIV - genetics; HIV - isolation & purification; Human immunodeficiency virus; Humans; Identification and classification; Influenza A; Influenza A virus - classification; Influenza A virus - genetics; Influenza A virus - isolation & purification; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Microscopy; Miniaturization; Multiplexing; Mutation; Nucleic acids; Pandemics; Pathogens; Physiological aspects; Reagents; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; SARS-CoV-2; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Virus Diseases - diagnosis; Virus Diseases - virology; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/s41586-020-2279-8

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens . Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health .