Title | Massively multiplexed nucleic acid detection with Cas13. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Ackerman, CM, Myhrvold, C, Thakku, SGowtham, Freije, CA, Metsky, HC, Yang, DK, Ye, SH, Boehm, CK, Kosoko-Thoroddsen, T-SF, Kehe, J, Nguyen, TG, Carter, A, Kulesa, A, Barnes, JR, Dugan, VG, Hung, DT, Blainey, PC, Sabeti, PC |
Journal | Nature |
Volume | 582 |
Issue | 7811 |
Pagination | 277-282 |
Date Published | 2020 Jun |
ISSN | 1476-4687 |
Keywords | Animals, Betacoronavirus, CRISPR-Associated Proteins, CRISPR-Cas Systems, Drug Resistance, Viral, Genome, Viral, HIV, Humans, Influenza A virus, Microfluidic Analytical Techniques, RNA, Guide, Kinetoplastida, SARS-CoV-2, Sensitivity and Specificity, Virus Diseases |
Abstract | <p>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.</p> |
DOI | 10.1038/s41586-020-2279-8 |
Alternate Journal | Nature |
PubMed ID | 32349121 |
PubMed Central ID | PMC7332423 |
Grant List | F32 CA236425 / CA / NCI NIH HHS / United States T32 GM087237 / GM / NIGMS NIH HHS / United States / HHMI / Howard Hughes Medical Institute / United States |