Journal
FUNGAL BIOLOGY
Volume 124, Issue 3-4, Pages 228-234Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.funbio.2020.02.006
Keywords
CRISPR/Cas9; Fungi genome editing; Off-site targets; Whole genome sequencing
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Funding
- European Research Council under the European Union's Horizon 2020 research and innovation program (consolidator grant conVIRgens) [771035]
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy [EXC2048/1, 390686111]
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The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing. (C) 2020 The Authors. Published by Elsevier Ltd on behalf of British Mycological Society.
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