A New Dual Luciferase Assay Using NanoLuc® Enables a Second Generation Coincidence Reporter System to Reduce False Hits in HTS Poster
Poster Oct 09, 2015
Christopher Eggers, Samuel Hasson, Brock Binkowski, Matt Robers, James Unch, Braeden Butler, , Keith Wood, James Inglese and Frank Fan
Luciferase-based reporter-gene assays remain a cornerstone of high-throughput screening of compounds because of their high sensitivity and dynamic range. However, a substantial number of non-relevant hits can be generated due to direct interaction of compounds with the luciferase reporter. To help differentiate compounds modulating the biological pathway of interest from those affecting the stability or activity of the reporter enzyme, we have developed a second-generation coincidence reporter system in which transcriptional activation leads to stoichiometric expression of two orthologous reporters that have dissimilar profiles of compound interference. In this system, firefly luciferase (Fluc) and PEST-destabilized NanoLuc® luciferase (NlucP) are expressed off the same promoter using ribosome skipping mediated by the P2A peptide.
To sensitively measure both Fluc and NanoLuc (Nluc) in the same sample, we have developed the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, a homogeneous lytic assay performed in an “add-read-add-read” format, in which the Fluc signal is quenched over a million-fold by addition of the Nluc reagent. The increased brightness of Nluc and improved Fluc inhibition means that Nluc can be detected at over 2-3 orders of magnitude lower molar concentration than Renilla luciferase in the existing homogenous firefly/Renilla dual-luciferase assay (Dual-Glo), allowing both luciferases to be dynamic reporters. Following single-copy integration of the Fluc-2A-NlucP biocircuit into a gene locus relevant to Parkinson’s disease, HTS using NanoDLR easily distinguished compounds affecting one of the reporters from those affecting transcription, yielding a >5-fold decrease in the number of hits.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE