We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of gene mutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.

我们之前报道了用于坏死性真菌植物病原体核盘菌的 CRISPR-Cas9 基因组编辑系统。该系统（TrpC-sgRNA 系统）基于 RNA 聚合酶 II (RNA Pol II) 启动子 (TrpC)在体内驱动 sgRNA 转录，成功地创建了基因插入突变体。然而，相对低效率的靶向基因编辑阻碍了该方法在核盘菌功能基因组研究中的应用. 为了进一步优化 CRISPR-Cas9 系统，无质粒 Cas9 蛋白/sgRNA 核糖核蛋白 (RNP) 介导的系统（RNP 系统）和基于质粒的 RNA 聚合酶 III 启动子（U6）驱动的 sgRNA 转录系统（U6- sgRNA 系统）的建立和评估。先前表征的草酰乙酸乙酰水解酶 ( Ssoah1 ) 基因座和编码聚酮化合物合酶 12 ( Sspks12 )的新基因座在本研究中成为目标，以创建功能缺失突变体。RNP 系统，类似于我们之前报道的 TrpC-sgRNA 系统，在Ssoah1基因位点以相当的效率产生突变。然而，这两个系统都没有成功地在Sspks12基因座。U6-sgRNA 系统 在两个位点都表现出显着更高的基因突变效率。该技术为靶向基因突变提供了一种简单有效的策略，从而将加快这一具有重要经济意义的植物病原体的致病性研究和发展步伐。