A Bioluminescence Resonance Energy Transfer-Based Reporter System: Characterization and Applications.

Abstract

Genome editing strategies and DNA repair research need powerful analytical tools. We generated a bioluminescence resonance energy transfer (BRET)-based reporter for the quantification of indel frequencies induced by DNA repair. The BRET reporter, expressed as a single molecule, consists of a mutated Renilla reniformis luciferase domain and a GFP2 domain separated by a shuttle-cloning box for the integration of any given endonuclease target sequence. The luciferase activity acts both as energy donor and as the internal standard, while the loss of GFP2 fluorescence acts as a reporter for the out-of-frame sequence alterations that result from the DNA repair via the non-homologous end joining/microhomology-mediated end joining DNA repair pathways of the endonuclease-mediated DNA double-strand break. This results in a decrease of the fluorescence/luminescence ratio. Employing this reporter in different experimental scenarios, using different cell lines and diseases targeted, we quantified the influence of both protein knockdown of DNA repair pathways as well as guide RNA mismatches on CRISPR-mediated nuclease activity and subsequent repair based on mutagenic repair on the reporter. In conclusion, we demonstrated this BRET-based reporter to be a robust and sensitive analytical tool for assessment of variety of different genome editing-based approaches.