(a) CRISPR-Cas13 RNA targeting complex components.
CRISPR-Cas13 enzymes are programmed by a crRNA, which is composed of a direct
repeat sequence (DR) flanked by a target complementary spacer sequence (shown in
blue). RNA cleavage is mediated by two Higher eukaryotic and prokaryotic
nuclease domains (HEPN) domains (shown as red boxes) within a typical Type VI-A
Cas13.
(b) Reporter unlocking via CRISPR-Cas13 collateral RNase
activity. The CRISPR-Cas13-RNA complex is activated by binding to a
complementary target RNA. The activation triggers collateral cleavage of a
nonspecific RNA sensor in trans. The fluorescently labeled
sensor is quenched when intact and emits fluorescence when cleaved by activated
CRISPR-Cas13 complex.
(c) SHERLOCK detection assay Schematic of SHERLOCK assay
steps, starting with pre-amplification of either a DNA or RNA target input.
Amplified targets are converted to RNA via T7 transcription and are then
detected by Cas13:crRNA complexes, which activate and cleave fluorescent RNA
sensors. For Cas12 detection, the T7 transcription step is omitted, allowing for
direct detection of amplified targets.