Pohl Carsten, Mózsik László, Driessen Arnold J M, Bovenberg Roel A L, Nygård Yvonne I
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
DSM Biotechnology Center, DSM Food Specialties B.V., Delft, The Netherlands.
Methods Mol Biol. 2018;1772:213-232. doi: 10.1007/978-1-4939-7795-6_12.
Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences . This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.
最近已经建立了几种CRISPR/Cas9工具，用于在多种丝状真菌中进行精确的基因组编辑。这个基因组编辑平台在靶点选择上具有高度灵活性，并且有可能在不引入转基因序列的情况下实现基因缺失。本章描述了一种用预组装的核糖核蛋白颗粒（RNP）转化产黄青霉原生质体的方法，该颗粒由纯化的Cas9蛋白和体外转录的单向导RNA（sgRNA）组成，用于删除基因组序列或用替代序列替换它们。这种方法有可能转移到所有能够获得原生质体的真菌菌株中。
