Head-to-Tail Multiplications of DNA Templates at Sites of Integration Mask Desired CRISPR/Cas9 - Mediated Genome Editing Events

CRISPR/Cas9 mediated genome editing has rapidly evolved into a key technology for precise gene editing in a wide range of model organisms, including mouse and human. Nowadays, CRISPR/Cas9 is widely applied for generation of animal models to investigate different human diseases. Despite the advantages of CRISPR/Cas9 based genome editing, a number of potential problems such as genome rearrangements and off target effects still impede the CRISPR/Cas9 technology for use in biomedical research and further efforts are necessary to overcome these hurdles.

Our study examines problems that affect direct knock-in genome targeting. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous 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 such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analyses of modified alleles are essential, and offer practical solutions to correctly identify precisely edited chromosomes.

Our findings are important to unlock the full potential of the CRISPR/Cas9-mediated genome editing protocols for the generation of custom designed gene variants for biomedical research and gene therapy.