A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
Fig 6
(a) Mechanism of action of Cas13a: the protein (blue) forms a complex with a crRNA (grey) that consists of a Cas13a-handle and a sequence complementary to the target RNA (orange). Upon binding of the target RNA to the Cas13a-crRNA complex, Cas13a undergoes a conformational change that activates promiscuous RNase activity: Cas13a becomes an unspecific RNase [31]. (b) Mechanism of detection of Cas13a activity: a short RNA strand is modified with a fluorophore and a quencher. Upon cleavage by Cas13a, the fluorophore is released from the proximity of the quencher, and therefore fluorescence increases. (c-d) Measurement of Cas13a activity with 100 nM target RNA on paper in the detector (c) or in bulk using a plate reader (d). Activity in presence of a cognate (i.e. complementary to the crRNA) RNA target is compared to a non-cognate target. Residual activity in the presence of non-cognate target is likely due to an unspecific activity of Cas13a. In (d), positive control in bulk contains RNase A. (e-f) Response of the assay to increasing concentrations of cognate or non-cognate RNA target, in the detector (e) or in bulk (f). The LOD and LOQ as calculated based on the mean and standard deviation of background measurements are shown with dotted lines. Thick line and shaded area (c) and dots and error bars (e) represent mean and measurement uncertainty as computed in S1 Appendix.