Luo Ning, Li Zeyu, Ling Jian, Zhao Jianlong, Li Yan, Yang Yuhong, Mao Zhenchuan, Xie Bingyan, Li Huixia, Jiao Yang
Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, China.
State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flower, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
J Fungi (Basel). 2023 May 19;9(5):595. doi: 10.3390/jof9050595.
is a filamentous fungus that serves as a biological control agent for multiple phytopathogens and as an important resource promising for fungicides. However, the lack of adequate knockout technologies has hindered gene function and biocontrol mechanism research of this species. This study obtained a genome assembly of T21, with a 41.4 Mb genome sequence comprising 8170 genes. Based on genomic information, we established a CRISPR/Cas9 system with dual sgRNAs targets and dual screening markers. CRISPR/Cas9 plasmid and donor DNA recombinant plasmid were constructed for disruption of the and genes. The result indicates the consistency between phenotypic characterization and molecular identification of the knockout strains. The knockout efficiencies of and were 100% and 89.1%, respectively. Moreover, sequencing revealed fragment deletions between dual sgRNA target sites or GFP gene insertions presented in knockout strains. The situations were caused by different DNA repair mechanisms, nonhomologous end joining (NHEJ), and homologous recombination (HR). Overall, we have successfully constructed an efficient and convenient CRISPR/Cas9 system in for the first time, which has important scientific significance and application value for studies on functional genomics of and other filamentous fungi.
是一种丝状真菌，可作为多种植物病原体的生物防治剂，也是一种有前景的重要杀真菌剂资源。然而，缺乏合适的基因敲除技术阻碍了该物种的基因功能和生物防治机制研究。本研究获得了T21的基因组组装结果，其基因组序列为41.4 Mb，包含8170个基因。基于基因组信息，我们建立了具有双sgRNA靶点和双筛选标记的CRISPR/Cas9系统。构建了CRISPR/Cas9质粒和供体DNA重组质粒，用于破坏和基因。结果表明敲除菌株的表型特征与分子鉴定结果一致。和的敲除效率分别为100%和89.1%。此外，测序显示敲除菌株中双sgRNA靶点之间存在片段缺失或GFP基因插入。这些情况是由不同的DNA修复机制，即非同源末端连接（NHEJ）和同源重组（HR）引起的。总体而言，我们首次在中成功构建了高效便捷的CRISPR/Cas9系统，这对和其他丝状真菌的功能基因组学研究具有重要的科学意义和应用价值。
