Massively multiplexed nucleic acid detection with Cas13

Details

• Alternative Title:
  Nature
• Personal Author:
  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.
• Description:
  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|.
• Subjects:
  Animals Article Betacoronavirus CRISPR-Associated Proteins CRISPR-Cas Systems Drug Resistance, Viral Genome, Viral HIV Humans Influenza A Virus Microfluidic Analytical Techniques RNA, Guide Sensitivity And Specificity Virus Diseases

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• Source:
  Nature. 582(7811):277-282
• Pubmed ID:
  32349121
• Pubmed Central ID:
  PMC7332423
• Document Type:
  Journal Article
• Funding:
  F32 CA236425/CA/NCI NIH HHS/United States ; HHMI/Howard Hughes Medical Institute/United States
• Volume:
  582
• Issue:
  7811
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:34935607b74cbf9ecbab22f076063dbf6e3827583c4904834b16abf2901fd3b1
• Download URL:
  https://stacks.cdc.gov/view/cdc/92508/cdc_92508_DS1.pdf
• File Type:
  [PDF - 25.14 MB ]