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

Nature Protocols volume 11, pages 598–615 (2016)Cite this article

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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.

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Figure 1: Outcomes of cellular repair upon designer nuclease–induced DSBs.
Figure 2: Theoretical results of GEF-dPCR.
Figure 3: Exemplary results of a GEF-dPCR.
Figure 4: Experimental workflow of GEF-dPCR.
Figure 5: Example of Sanger sequencing results to characterize DN-induced mutation pattern and to facilitate subsequent probe design.
Figure 6: DN-induced mutation pattern in different cell types as visualized by NGS.
Figure 7: Analysis of GEF-dPCR data.
Figure 8: Optimization of GEF-dPCR experiments.

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Acknowledgements

The authors are indebted to T. Sonntag for excellent technical support. In addition, we thank T. Stahl and A. Badbaran for the very valuable discussion. We are very grateful to K. Riecken for critically reading the manuscript and W. Qasim for supporting the generation of the manuscript. This work has been supported by the Deutsche Forschungsgemeinschaft (DFG; SFB841/SP2 to B.F.), the National Institute of Health Research via the Great Ormond Street/Institute of Child Health Biomedical Research Centre (to U.M.) and the German Centre for Infection Research (DZIF).

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  1. Ulrike Mock

    Present address: Present address: Molecular and Cellular Immunology Unit, Institute of Child Health, University College London (UCL), London, UK.,

Authors and Affiliations

  1. Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre, Hamburg-Eppendorf (UKE,

    Ulrike Mock & Boris Fehse

  2. ), Hamburg, Germany

    Ulrike Mock & Boris Fehse

  3. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany

    Ilona Hauber

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Contributions

U.M. and B.F. developed the technique, performed most experiments, analyzed and interpreted data and wrote the manuscript. I.H. performed the in vivo experiment. All authors have read the manuscript and confirmed their authorship.

Corresponding authors

Correspondence to Ulrike Mock or Boris Fehse.

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Competing interests

U.M. and B.F. have submitted a patent application (decision pending) for the exemplarily used designer nuclease (CCR5-Uco-TALEN). They have no competing interests regarding the described PCR method. I.H. has no competing financial interests.

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Mock, U., Hauber, I. & Fehse, B. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11, 598–615 (2016). https://doi.org/10.1038/nprot.2016.027

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