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 (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.
Highlights
Repairing DNA damage plays a key role in maintaining genome integrity and cell viability
The bioluminescent repair reporter (BLRR) consists of secreted Gaussia luciferase (Gluc) and Vargula luciferase (Vluc) for simultaneous monitoring of homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively
We observed reduced DNA-PKcs expression following B02 treatment, which likely resulted in the decreased Vluc signals, especially at higher dosages (Supplementary Figure 7C). These results indicate that BLRR signals and the BLRR ratio can be applied to investigate the effect of small molecules or other modalities in modulating double strand break (DSB) repair, which is of relevance to high-throughput screening and preclinical studies
Summary
Repairing DNA damage plays a key role in maintaining genome integrity and cell viability. Much effort in gene therapy development has focused on enhancing HDR over NHEJ during DSB repair to introduce functional genes, either by controlling genome editing tools, the cell cycle[11,12], optimising donor templates[13], or using small molecules to inhibit NHEJ-related proteins[14,15,16]. Investigating DSB repair outcomes can be time-consuming, and typically requires disruption of cells for subsequent DNA sequence analyses This challenge has impeded high-throughput HDR optimisation for the development of cancer and gene therapies[3]. Vluc activity remained similar between CAY-treated and dimethyl sulphoxide (DMSO) controls
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
