Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo

• Published in: Nature protocols Vol. 16; no. 8; pp. 3933 - 3953
• Main Authors: Chien, Jasper Che-Yung, Badr, Christian E., Lai, Charles Pin-Kuang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.08.2021; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/245/2222; 631/337/1427/2122; Analytical Chemistry; Animals; Assaying; Biochemical assays; Biological Techniques; Bioluminescence; Biomedical and Life Sciences; Cell Line; Complementation; Computational Biology/Bioinformatics; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA repair; DNA Repair - physiology; DNA sequencing; Double-strand break repair; Editing; Gaussia; Genetic research; Genetically modified organisms; Genome editing; Genomes; Homology; Humans; Life Sciences; Luminescent Measurements - methods; Marking and tracking techniques; Mice; Mice, Transgenic; Microarrays; Modulators; Molecular biology; Neoplasms, Experimental; Next-generation sequencing; Noninvasive evaluation; Nuclease; Organic Chemistry; Protocol; Repair; Tracking; Vargula; Yeast; Taiwan
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-021-00564-8

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. This protocol sets up 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.