Predicting the mutations generated by repair of Cas9-induced double-strand breaks
| Autor: | Yaron Galanty, Vitalii Kleshchevnikov, Andrew R. Bassett, Alexander Strong, Luca Crepaldi, Anton Khodak, Heather P. Harding, Pietro De Angeli, Clara Alsinet, Vladimir Yu. Kiselev, Felicity Allen, Stephen P. Jackson, Francisco Muñoz-Martínez, Petra Páleníková, Michael Kosicki, Leopold Parts, Emmanouil Metzakopian |
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| Rok vydání: | 2018 |
| Předmět: |
Double strand
0303 health sciences Cas9 Biomedical Engineering Bioengineering Computational biology Biology Applied Microbiology and Biotechnology Phenotype Article DNA sequencing Dna mutation 03 medical and health sciences 0302 clinical medicine Genome editing Molecular Medicine Guide RNA 030217 neurology & neurosurgery 030304 developmental biology Biotechnology Sequence (medicine) |
| Zdroj: | Nature Biotechnology. 37:64-72 |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/nbt.4317 |
| Popis: | The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments. |
| Databáze: | OpenAIRE |
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