Massively multiplexed nucleic acid detection with Cas13

Citation:

Cheri M Ackerman*, Cameron Myhrvold*†, Sri Gowtham Thakku, Catherine A Freije, Hayden C Metsky, David K Yang, Simon H Ye, Chloe K Boehm, Tinna-Sólveig F Kosoko-Thoroddsen, Jared Kehe, Tien G Nguyen, Amber Carter, Anthony Kulesa, John R Barnes, Vivien G Dugan, Deborah T Hung, Paul C Blainey*†, and Pardis C Sabeti*. 2020. “Massively multiplexed nucleic acid detection with Cas13.” Nature, 582, 7811, Pp. 277-282.

ISSN:

1476-4687

Abstract:

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.

DOI:

10.1038/s41586-020-2279-8

Alternate Journal:

Nature
Last updated on 07/30/2020