Extended Data Fig. 4: LbuCas13a-EiCsm6 detection assay with unmodified and single-fluoro activators at high target RNA concentrations, related to Fig. 2.
From: Accelerated RNA detection using tandem CRISPR nucleases
a) Detection assay using LbuCas13a complexed with two crRNAs, 604 and 612, and EiCsm6 with a A6-U5 oligonucleotide activator22. An in vitro transcribed RNA corresponding to a fragment of the SARS-CoV-2 genome (gblock) was added at concentrations ranging from 1.25 × 106 copies per µl (cp/µl) of RNA (2 pM) to 1.25 × 108 cp/µl) of RNA (200 pM). Control reactions that lack both target RNA and the EiCsm6 activator, or contain only the fluorescent reporter (Reporter only) are shown for comparison. Mean normalized fluorescence (F/F0) and s.e.m (n = 3) are plotted as lines with error bars over the reaction time course. A schematic of the A6-U5 activator is shown above the graph. b) As in a but using a single-fluoro A6-U5 oligonucleotide as an EiCsm6 activator. The adenosine bearing a 2´-F modification is colored pink in the schematic above the graph. Controls from a that lack both target RNA and EiCsm6 activator, or contain only the reporter are overlaid in the graph, as all experiments in this figure were run in parallel.
