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Massively multiplexed nucleic acid detection with Cas13.
Nature. 2020 Jun; 582(7811):277-282.Nat

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[1-3] while simultaneously testing for many pathogens[4-6]. 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[7] self-organize in a microwell array[8] 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[9-11].

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Authors+Show Affiliations

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Biological Engineering, MIT, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. cmyhrvol@broadinstitute.org. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. cmyhrvol@broadinstitute.org.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Division of Health Sciences and Technology, Harvard Medical School and MIT, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Ph.D. Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Division of Health Sciences and Technology, Harvard Medical School and MIT, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.

Kosoko-Thoroddsen TF

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Biological Engineering, MIT, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Biological Engineering, MIT, Cambridge, MA, USA.

Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA.

Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Molecular Biology Department and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. pblainey@broadinstitute.org. Department of Biological Engineering, MIT, Cambridge, MA, USA. pblainey@broadinstitute.org. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA. pblainey@broadinstitute.org.

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. Howard Hughes Medical Institute, Chevy Chase, MD, USA. Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

MeSH

AnimalsBetacoronavirusCRISPR-Associated ProteinsCRISPR-Cas SystemsDrug Resistance, ViralGenome, ViralHIVHumansInfluenza A virusMicrofluidic Analytical TechniquesRNA, Guide, CRISPR-Cas SystemsSARS-CoV-2Sensitivity and SpecificityVirus Diseases

Pub Type(s)

Journal Article

Research Support, N.I.H., Extramural

Research Support, Non-U.S. Gov't

Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

32349121

Citation

Ackerman, Cheri M., et al. "Massively Multiplexed Nucleic Acid Detection With Cas13." Nature, vol. 582, no. 7811, 2020, pp. 277-282.

Ackerman CM, Myhrvold C, Thakku SG, et al. Massively multiplexed nucleic acid detection with Cas13. Nature. 2020;582(7811):277-282.

Ackerman, C. M., Myhrvold, C., Thakku, S. G., Freije, C. A., Metsky, H. C., Yang, D. K., Ye, S. H., Boehm, C. K., Kosoko-Thoroddsen, T. F., Kehe, J., Nguyen, T. G., Carter, A., Kulesa, A., Barnes, J. R., Dugan, V. G., Hung, D. T., Blainey, P. C., & Sabeti, P. C. (2020). Massively multiplexed nucleic acid detection with Cas13. Nature, 582(7811), 277-282. https://doi.org/10.1038/s41586-020-2279-8

Ackerman CM, et al. Massively Multiplexed Nucleic Acid Detection With Cas13. Nature. 2020;582(7811):277-282. PubMed PMID: 32349121.

* Article titles in AMA citation format should be in sentence-case

TY - JOUR T1 - Massively multiplexed nucleic acid detection with Cas13. AU - Ackerman,Cheri M, AU - Myhrvold,Cameron, AU - Thakku,Sri Gowtham, AU - Freije,Catherine A, AU - Metsky,Hayden C, AU - Yang,David K, AU - Ye,Simon H, AU - Boehm,Chloe K, AU - Kosoko-Thoroddsen,Tinna-Sólveig F, AU - Kehe,Jared, AU - Nguyen,Tien G, AU - Carter,Amber, AU - Kulesa,Anthony, AU - Barnes,John R, AU - Dugan,Vivien G, AU - Hung,Deborah T, AU - Blainey,Paul C, AU - Sabeti,Pardis C, Y1 - 2020/04/29/ PY - 2019/03/20/received PY - 2020/04/20/accepted PY - 2020/4/30/pubmed PY - 2020/6/19/medline PY - 2020/4/30/entrez SP - 277 EP - 282 JF - Nature JO - Nature VL - 582 IS - 7811 N2 - 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[1-3] while simultaneously testing for many pathogens[4-6]. 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[7] self-organize in a microwell array[8] 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[9-11]. SN - 1476-4687 UR - https://www.unboundmedicine.com/medline/citation/32349121/Massively_multiplexed_nucleic_acid_detection_using_Cas13. DB - PRIME DP - Unbound Medicine ER -