RNaseAlert Readout
To characterize our Cas13a, we first turned to the standard of the field, namely the RNase Alert detection kit. This was used by Gootenberg and Doudna to characterize the Cas13a and detect pathogen RNA sequences1. In the absence of Cas13a activation, the physical proximity of the quencher dampens fluorescence from the Fluor and there is no fluorescence activity. When Cas13a is activated, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution, emitting a bright green signal when excited by light of the appropriate wavelength. We did most of our experiments using the RNaseAlert system and the corresponding results are in the Cas13a and target subsections.
The fluorescence signal quality of the RNaseAlert assay was very good, however one cannot use this readout system without the fluorescence detector. Also, since the RNaseAlert is a modified RNA, it is somehow expensive in comparison to other readouts.
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