Muñoz-Fernández Gloria, Jiménez Alberto, Revuelta José Luis
Metabolic Engineering Group, Department of Microbiology and Genetics, University of Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
Bio Protoc. 2020 Jun 20;10(12):e3660. doi: 10.21769/BioProtoc.3660.
The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of . The filamentous fungus is currently used for the industrial production of riboflavin (vitamina B2). In addition, produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 system comprises an RNA guided DNA endonuclease (Cas9) and a guide RNA (gRNA), which is complementary to the genomic target region. The Cas9 nuclease requires a 5'-NGG-3' trinucleotide, called protospacer adjacent motif (PAM), to generate a double-strand break (DSB) in the genomic target, which can be repaired with a synthetic mutagenic donor DNA (dDNA) by homologous recombination (HR), thus introducing a specific designed mutation. The CRISPR/Cas9 system adapted for largely facilitates the genomic edition of this industrial fungus.
CRISPR/Cas9系统是一种新型基因工具，可对几乎任何基因组序列进行精确操作。在本方案中，我们使用特定的CRISPR/Cas9系统来操作……丝状真菌目前用于工业生产核黄素（维生素B2）。此外，……还能产生其他高价值化合物，如叶酸、核苷和生物脂质。有一个大型分子工具箱可用于该真菌的基因组操作，包括基因靶向方法、异源表达模块的快速组装，以及最近适用于……的单载体CRISPR/Cas9编辑系统，该系统允许实施无标记工程策略。CRISPR/Cas9系统由RNA引导的DNA内切酶（Cas9）和与基因组靶区域互补的引导RNA（gRNA）组成。Cas9核酸酶需要一个5'-NGG-3'三核苷酸，称为原间隔相邻基序（PAM），以在基因组靶标中产生双链断裂（DSB），该双链断裂可通过同源重组（HR）用合成诱变供体DNA（dDNA）修复，从而引入特定设计的突变。适用于……的CRISPR/Cas9系统极大地促进了这种工业真菌的基因组编辑。
