Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases

• Published in: Nature protocols Vol. 11; no. 3; pp. 598 - 615
• Main Authors: Mock, Ulrike, Hauber, Ilona, Fehse, Boris
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.03.2016; Nature Publishing Group
• Subjects: 42/109; 42/41; 631/1647/1513/1967/2316; 631/1647/1513/1967/3196; 631/1647/1513/2216; 631/61/191/1908; Alleles; Analytical Chemistry; Base Sequence; Biological Techniques; Cell Line; Computational Biology/Bioinformatics; CRISPR; CRISPR-Cas Systems; DNA - genetics; DNA End-Joining Repair; DNA probes; DNA Probes - genetics; Endonucleases - genetics; Endonucleases - metabolism; Genetic engineering; Genetic Engineering - methods; Genetic modification; Genome editing; Genomes; Humans; In vivo methods and tests; Innovations; Life Sciences; Methods; Microarrays; Molecular Sequence Data; Mutation; New technology; Non-homologous end joining; Nuclease; Nucleases; Organic Chemistry; Polymerase chain reaction; Polymerase Chain Reaction - methods; Probes; Properties; protocol; Target detection; Transcription; Transcription activator-like effector nucleases
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.027

Genome editing using designer nucleases such as TALENs or the CRISPR-Cas9 system is hampered by a lack of methods to detect and quantify the products. Here the authors present GEF-dPCR, a droplet-based digital PCR method for assessing gene-editing frequencies. Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo , e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.