Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5´-phosphate decarboxylase involved in pyrimidine biosynthesis and the α-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.

Claviceps purpurea是一种众所周知的丝状真菌，是一种广泛存在的植物病原体，但它也是制药业开发的重要麦角生物碱生产商。在这项工作中，我们证明了 CRISPR/Cas9 可以作为一种工具，用于在C. purpurea 中靶向pyr4和TrpE进行定向诱变分别编码参与嘧啶生物合成的乳清酸 5'-磷酸脱羧酶和参与色氨酸生物合成的邻氨基苯甲酸合酶的 α-亚基的基因。在原生质体转化和单孢子分离后，选择显示尿苷或色氨酸营养缺陷的同核突变体。在所有情况下，插入或插入与缺失相结合主要在 PAM 序列上游 3 bp 处发现。然而，与同源重组介导的TrpE基因敲除相比，CRISPR/Cas9 和 CRISPR/Cas9 介导的同源定向修复的转化效率仅略有提高。此外，色氨酸营养缺陷型对黑麦植物没有感染性，这可能是由于植物激素生长素的产生减少，生长素是由C. purpurea来自 indole-3-glycerolphosphate 在 Trp 依赖性和 Trp 非依赖性生物合成途径中，并帮助真菌定殖植物宿主。已经证实，包含自主复制序列的AMA1 CRISPR / Cas9载体可通过的进一步培养完全除去C.菊在非选择性培养基中。该方法能够在Claviceps 中引入多个突变，并使工业菌株的代谢工程可行。