Journal
SCIENCE
Volume 360, Issue 6387, Pages 439-+Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.aaq0179
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Funding
- Paul and Daisy Soros Fellowship
- NIH F30 National Research Service Award [1F30-CA210382]
- Defense Threat Reduction Agency [HDTRA1-14-1-0006]
- Paul G. Allen Frontiers Group
- Wyss Institute
- NIH [1R01-HG009761, 1R01-MH110049, 1DP1-HL141201]
- Howard Hughes Medical Institute
- New York Stem Cell Foundation
- Simons Foundation
- Paul G. Allen Family Foundation
- Vallee Foundation
- Poitras Center for Affective Disorders Research at MIT
- Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT
- Skolkovo Institute of Science and Technology
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Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
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