背景:指状青霉是一种真菌植物病原体,可导致柑橘类水果的绿色霉菌病。由于其经济相关性,许多努力都集中在开发这种真菌的基因工程工具上。CRISPR/Cas9技术的适应以前是通过基于AMA1的自我复制质粒实现的,用于无标记基因编辑。但由此产生的效率(10%)限制了其实际实施。在这项研究中,我们的目标是提高CRISPR/Cas9介导的基因编辑在指毛中的效率,以促进其实际应用.
结果:通过在促进较慢生长速率的培养基中在选择条件下进行额外的培养条纹来增加培养时间,显着提高了指状品中的基因编辑效率高达54-83%。为了证明这一点,我们破坏了5个候选基因,这些基因是根据我们之前的高通量基因表达研究选择的,这些研究旨在阐明洋地黄对抗真菌蛋白PdAfpB的转录组反应.其中两个基因导致视觉表型变化(PDIG_53730/pksP,和PDIG_54100/arp2),并允许启动协议优化。其他三个候选物(PDIG_56860、PDIG_33760/rodA和PDIG_68680/dfg5)没有视觉上相关的表型,并且被靶向以确认方案的高效率。
结论:通过选择方法的修改,洋地黄的基因组编辑效率从10%显着提高到83%,这证明了CRISPR/Cas9系统在这种植物病原真菌中进行基因破坏的可行性。此外,本研究中描述的方法可能有助于提高其他经济相关真菌物种的CRISPR/Cas9基因编辑效率,而这些真菌物种通过CRISPR/Cas9进行编辑的效率仍然很低.
BACKGROUND: Penicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use.
RESULTS: Increasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54-83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol.
CONCLUSIONS: Genome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.