A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo. Issue 17 (14th August 2020)

• Record Type: Journal Article
• Authors: Chien, Jasper Che-Yung; Tabet, Elie; Pinkham, Kelsey; da Hora, Cintia Carla; Chang, Jason Cheng-Yu; Lin, Steven; Badr, Christian E; Lai, Charles Pin-Kuang
• Is Part Of: Nucleic acids research. Volume 48:Issue 17(2020)
• Page Start: e100
• Page End: e100
• Publication Date: 2020-08-14
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa669
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR ( R 2 = 0.9722) and NHEJ ( R 2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo . Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1–10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.