title: Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.
abstract: 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.
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre, Hamburg-Eppendorf (UKE), Hamburg, Germany.
authors.forename: Ulrike
authors.identifier: http://orcid.org/0000-0003-2961-5327
authors.initials: U
authors.lastname: Mock
authors.affiliation: Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
authors.forename: Ilona
authors.initials: I
authors.lastname: Hauber
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre, Hamburg-Eppendorf (UKE), Hamburg, Germany.
authors.forename: Boris
authors.identifier: http://orcid.org/0000-0001-9780-7211
authors.initials: B
authors.lastname: Fehse
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004247:DNA / Q000235:genetics; D059766:DNA End-Joining Repair; D015342:DNA Probes / Q000235:genetics; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D015342:DNA Probes; D004247:DNA; D004720:Endonucleases