Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.
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.
|Year of Publication||
2018 04 27
|PubMed Central ID||
R01 MH110049 / MH / NIMH NIH HHS / United States
HHMI / Howard Hughes Medical Institute / United States
DP1 HL141201 / HL / NHLBI NIH HHS / United States
R01 HG009761 / HG / NHGRI NIH HHS / United States
RM1 HG006193 / HG / NHGRI NIH HHS / United States
F30 CA210382 / CA / NCI NIH HHS / United States