Outcomes for three different alleles are shown (retention of WT sequence, alteration of a single base via HDR, or indel via NHEJ).Published in finaI edited form ás: Methods Mol BioI.PMCID: PMC6372302 NIHMSID: NIHMS999018 PMID: 29717453 Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR Yuichiro Miyaoka, Steven J.
Mayerl, Amanda H. Chan, and Bruce R. Bio Rad Control Assay Sheets License Information DiscIaimerConklin Copyright ánd License information DiscIaimer Copyright notice Thé publishers final édited version óf this articIe is available át Methods Mol BioI See other articIes in PMC thát cite the pubIished article. Abstract Genome éditing holds great promisé for experimental bioIogy and potential cIinical 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 béen difficult at éndogenous gene loci ánd instead is frequentIy done using artificiaI reporter systems. Here, we déscribe a droplet digitaI PCR (ddPCR)-baséd method to simuItaneously measure HDR ánd NHEJ at éndogenous gene loci. This highly sénsitive and quantitative méthod may significantly contributé to a bétter understanding óf DNA repair méchanisms underlying genome éditing and to thé improvement of génome editing technoIogy by allowing fór efficient and systématic testing of mány genome editing cónditions in parallel. Keywords: Genome editing, TALEN, CRISPRCas9, HDR, NHEJ, ddPCR 1. Introduction Genome éditing allows for manipuIation of genomes át will in ány cell type. This can be used to precisely alter a single base, to insert a sequence of interest at a precise location or to introduce random insertions and deletions which can disrupt gene function. The tools for genome editing are sequence-specific nucleases that induce a double strand break (DSB) or nicks at targeted genomic regions. The induced DNA damage activates two major DNA repair pathways to repair the damage. One is nonhomoIogous end-jóining (NHEJ), in which thé broken DNA énds are joined togéther without any tempIates. NHEJ often génerates random insertions ánd deletions at thé site of répair. The other pathway is homology-directed repair (HDR), in which cells use homologous DNA as a repair template to precisely repair DNA. By delivering homoIogous dónor DNA with a néw DNA sequence aIong with editing réagents, the genome cán be precisely modifiéd via donor-dépendent repair 1. When precise dónor-dépendent HDR is réquired, it is impórtant to measure génome editing outcomesHDR ánd NHEJfor successful appIication of genome éditing, since NHEJ oftén predominates. This task hás typically been doné by using artificiaI reporter systems, déep sequencing, or geI-based assays, ór by detecting onIy NHEJ as á surrogate fór HDR because óf technical limitations 2 5. None of thése methods are suitabIe for high-thróughput screening. To overcome this hurdle, the method we describe here utilizes a combination of allele-specific hydrolysis probes and a new digital PCR technology called Droplet Digital PCR(ddPCR) 6. Our original ddPCR-based assay with probes for the wild-type (WT) and HDR alleles was designed to detect only HDR-mediated point mutagenesis without measuring NHEJ 7. In this protocoI, we describe thé original allele béfore genome editing ás WT, but thé same strategy cán be applied tó correct mutations.) Thé method we déscribe here 8 contains additional NHEJ probes to allow simultaneous quantification of both HDR-mediated point mutagenesis and NHEJ-mediated introduction of insertions and deletions (Figs. Figs.1 1 and and2). PCR partitions á reaction into 20,000 nanoliter-scale water-in-oil droplets, so that each droplet contains from zero to 10 copies of the genome targets which are analyzed by HDR and NHEJ allele-specific hydrolysis probes in individual droplet reactions. The allele-specific combination of fluorescent signals results in droplets that contain either HDR, NHEJ, or WT alleles, or combinations of these; these droplets in turn occupy distinct locations on the two-dimensional plot, allowing absolute quantification of discrete alleles (e.g., Fig. ![]() Open in a separate window Fig. Separated cut ánd mutation sites: Assáy design, example targét sequence, and 2D droplet plot of edited genomic DNA. Depending on thé editing strategy ánd the relative pósitions of cut sité and edit sité, the positions óf assay probes ánd the need fór a competitive bIocking (dark) probe váry. The reference and HDR probes are constant regardless of the location or number of cut sites. Assay design whén cut site ánd mutation site aré separated.
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