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Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method

文献类型： 外文期刊

作者： Yu, Lu 1 ; Xiao, Meili 1 ; Zhu, Zhihua 1 ; Wang, Yinmei 1 ; Zhou, Zhihua 1 ; Wang, Pingping 1 ; Zou, Gen 1 ;

作者机构： 1.Chinese Acad Sci, CAS Ctr Excellence Mol Plant Sci, Inst Plant Physiol & Ecol, CAS Key Lab Synthet Biol, 300 Fenglin Rd, Shanghai 200032, Peoples R China

2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China

3.Shanghai Acad Agr Sci, Inst Edible Fungi, Shanghai Key Lab Agr Genet & Breeding, 1000 Jinqi Rd, Shanghai 201403, Peoples R China

关键词： Ribonucleoprotein; Genome editing; Ergot alkaloids; Biosynthetic pathway; Homologous recombination

期刊名称：SYNTHETIC AND SYSTEMS BIOTECHNOLOGY （ 影响因子：4.692； 五年影响因子：5.23 ）

ISSN： 2405-805X

年卷期： 2022 年 7 卷 2 期

页码：

收录情况： SCI

摘要： Claviceps purpurea produces many pharmacologically important ergot alkaloids (EAS), which are widely used to treat migraine and hypertension and to aid childbirth. Although an EAS biosynthetic cluster of C. purpurea has been discovered more than 20 years ago, the complete biosynthetic pathway of EAS has not been fully characterized until now. The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C. purpurea. The conventional gene manipulation method for C. purpurea relies on homologous recombination (HR), although the efficiency of HR in C. purpurea is very low (similar to 1-5%). Consequently, the disruption of target genes is laborious and time-consuming. Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently, their gene-disruption efficiency is still very low. Here, we developed an efficient genome-editing system in C. purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes. As proof of principle, three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system, with editing efficiencies ranging from 50% to 100%. Inactivation of the three genes, which are closely related to uridine biosynthesis (ura5), hypha morphology (rac), and EAS production (easA), resulted in a uridine auxotrophic mutant, a mutant with a drastically different phenotype in axenic culture, and a mutant that did not produce EAS, respectively. Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C. purpurea, which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C. purpurea.

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