A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo

Abstract

<jats:p>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 (R2 = 0.9722) and NHEJ (R2 = 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.</jats:p>

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“…Treatment with YTX-7739 also led to a decreased RAD51 expression concomitant with an upregulation of the DNA damage marker γ-H2AX ( Figure 2B ). We used an HDR/NHEJ reporter system 23 and confirmed that treatment of GSCs with YTX-7739 significantly decreases the ratio of HDR/NHEJ ( Figure 2F ), in line with RAD51 downregulation. Next, we asked whether sensitization to TMZ or RT could occur in GSCs resistant to the SCD inhibitor or those resistant to TMZ.…”

“…The BLRR system 23 was used to track DNA damage repair through Homology directed repair (HDR) and non-homologous end-joining (NHEJ) as previously described 64 . In brief, GSCs were sequentially transduced to express the three components of this reporter: BLRR, trGluc, and I-SceI.…”