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.

中文翻译：

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Cas9HF1 增强了黑粉病菌的特异性

成簇的规则间隔短回文重复序列 (CRISPR)-Cas9 系统被广泛用作精确操纵 sgRNA（单向导 RNA）靶向的基因组序列的工具，并适用于不同物种的基因组编辑。CRISPR-Cas9 的主要问题之一是脱靶效应的可能性，这可以通过部署高保真 Cas9 变体来解决。Ustilago maydis 是一种玉米真菌病原体，几十年来一直是生物营养病原体的模式生物。CRISPR-Cas9 在 U. maydis 中的成功应用极大地促进了效应生物学研究。在这里，我们构建了一个 U. maydis 报告菌株，它允许在体内量化三种高保真 Cas9 变体 Cas9HF1、Cas9esp1.1 和 Cas9hypa 的效率和目标特异性。这种方法将 Cas9HF1 鉴定为 U. maydis 中最具体的 Cas9 变体。此外，全基因组测序显示通过 CRISPR-Cas9 编辑在美国梅迪斯木中不存在脱靶效应。