Abstract
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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Acknowledgements
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.
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C.P.-K.L. and C.E.B. conceived and designed the study. J.C.C. conducted the in vitro experiments and developed the procedures. C.E.B developed the in vivo procedures. The manuscript was written by J.C.C., C.E.B. and C.P.-K.L. with input from all authors.
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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
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DOI: https://doi.org/10.1038/s41596-021-00564-8
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