Massively multiplexed nucleic acid detection with Cas13.

TitleMassively multiplexed nucleic acid detection with Cas13.
Publication TypeJournal Article
Year of Publication2020
AuthorsAckerman, 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
Date Published2020 Jun
KeywordsAnimals, 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

<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>

Alternate JournalNature
PubMed ID32349121
PubMed Central IDPMC7332423
Grant ListF32 CA236425 / CA / NCI NIH HHS / United States
T32 GM087237 / GM / NIGMS NIH HHS / United States
/ HHMI / Howard Hughes Medical Institute / United States