Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
STAR Protoc. 2021 Dec 24;3(1):101072. doi: 10.1016/j.xpro.2021.101072. eCollection 2022 Mar 18.
Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using (synonym of ), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021).
通过同源重组 (HR) 进行基因替换已广泛用于功能基因组研究。然而,丝状真菌中 HR 修复的总体效率可能较低,且该过程繁琐。在这里,我们提供了一种详细的方案,通过 Cas12a 核糖核蛋白 (RNP) 介导的 DNA 双链断裂将供体 DNA 插入感兴趣区域,从而实现高效的基因编辑。我们使用 (同义名 ), 一种用于研究植物-真菌相互作用的模式植物病原真菌来演示该方案。有关该方案使用和执行的完整详细信息,请参阅 Huang 等人 (2021)。