Abstract
Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.
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Bio-Rad Bulletin 6407, Droplet Digital Applications guide
Acknowledgment
We thank Jennifer R. Berman, Samantha B. Cooper, Bin Zhang, and George A. Karlin-Neumann (Bio-Rad) for technical help and helpful discussions. This work was supported by the National Institutes of Health (U01-HL100406, U01-GM09614, R01-HL108677, U01-HL098179, U01-HL099997, P01-HL089707, and R01-HL060664 to B.R.C.); the UCSF Liver Center to B.R.C., the Bluefield Project to Cure Frontotemporal Dementia to B.R.C., the Uehara Memorial Foundation Research Fellowship to Y.M., and Gladstone-CIRM Fellowship to Y.M.
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Miyaoka, Y., Mayerl, S.J., Chan, A.H., Conklin, B.R. (2018). Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_20
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_20
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