Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events
| Autor: | Cord Sunderkötter, Roland Wedlich-Söldner, Juergen Brosius, Hermann Pavenstädt, Yuri B. Schwartz, Thomas Pap, Boris V. Skryabin, Sven G. Meuth, Helena Kaiser, Anja Stegemann, Johannes Roth, J. Sherwood, Delf-Magnus Kummerfeld, Timofey S. Rozhdestvensky, Birte Seeger, Leonid Gubar |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Male
DNA repair Computer science Cell- och molekylärbiologi ved/biology.organism_classification_rank.species Computational biology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing Conditional gene knockout Genetics CRISPR Animals Allele Model organism Molecular Biology Research Articles Crosses Genetic 030304 developmental biology Sequence (medicine) Gene Editing Mice Knockout 0303 health sciences Multidisciplinary Genome Cas9 ved/biology SciAdv r-articles DNA Templates Genetic Non-homologous end joining Mice Inbred C57BL chemistry Genetic Loci Gene Targeting Models Animal Female CRISPR-Cas Systems 030217 neurology & neurosurgery Cell and Molecular Biology Research Article |
| Zdroj: | Science Advances |
| ISSN: | 2375-2548 |
| Popis: | Knock-in genome targeting risks: Comprehensive locus analysis is essential for precision chromosome-editing identification. 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. |
| Databáze: | OpenAIRE |
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