The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2–4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.
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We thank members of the Lai Lab and the Badr Lab for the very helpful discussions. This work was supported by the Ministry of Science and Technology (MOST) grants (104-2320-B-007-005-MY2, 106-2320-B-007-004-MY3, 109-2628-B-001-017 to C.P.-K.L.), Academia Sinica Innovative Materials and Analysis Technology Exploration (i-MATE) Program (AS-iMATE-107-33 to C.P.-K.L.), Academia Sinica Career Development Award (AS-CDA-109-M04 C.P.-K.L.), the National Institutes of Health (K22CA197053 and R01NS113822 to C.E.B.) and the American Brain Tumor Association (ABTA) Discovery Grant supported by the Uncle Kory Foundation to C.E.B.
A provisional patent application on the BLRR reporter has been submitted.
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Key reference using this protocol
Chien, J. C. et al. Nucleic Acids Res. 48, e100 (2020): https://doi.org/10.1093/nar/gkaa669
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Chien, J.CY., Badr, C.E. & Lai, C.PK. Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo. Nat Protoc 16, 3933–3953 (2021). https://doi.org/10.1038/s41596-021-00564-8