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Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15.

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.

Gootenberg JS1,2,3,4,5, Abudayyeh OO1,2,3,4,6, Kellner MJ1, Joung J1,2,3,4, Collins JJ1,4,6,7,8, Zhang F9,2,3,4.

Author information

1
Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA.
2
McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA.
3
Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA.
4
Department of Biological Engineering, MIT, Cambridge, MA 02139, USA.
5
Department of Systems Biology, Harvard University, Boston, MA 02115, USA.
6
Department of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA.
7
Institute for Medical Engineering and Science, MIT, Cambridge, MA 02139, USA.
8
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
9
Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA. zhang@broadinstitute.org.

Abstract

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.

PMID:
29449508
PMCID:
PMC5961727
DOI:
10.1126/science.aaq0179
[Indexed for MEDLINE]
Free PMC Article

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