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.