Journal or Publishing Institution: Science Advances
Date of Publication: 02/12/2020
Study: https://advances.sciencemag.org/content/6/7/eaax2941
Author(s): Skryabin, B.V., Kummerfeld, D.M., Gubar, L., Seeger, B., Kaiser, H., Stegemann, A., Roth, J., Meuth, S.G., Pavenstädt, H., Sherwood, J. and Pap, T.
Article Type: Peer Reviewed Study
Abstract:
CRISPR-Cas9–mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.
Keywords: CRISPR-Cas9, genome editing, DNA repair, PCR analysis, mice, knockout models, DNA insertion
Citation:
Skryabin, B.V., Kummerfeld, D.M., Gubar, L., Seeger, B., Kaiser, H., Stegemann, A., Roth, J., Meuth, S.G., Pavenstädt, H., Sherwood, J. and Pap, T., 2020. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events. Science Advances, 6(7), p.eaax2941.
Category:
- Health effects
- Environmental effects
- Regulatory issues
Record ID: 2533
