Ropero-Pérez Carolina, Marcos Jose F, Manzanares Paloma, Garrigues Sandra
Food Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Catedrático Agustín Escardino Benlloch 7, Paterna, Valencia, 46980, Spain.
Fungal Biol Biotechnol. 2024 Jul 13;11(1):8. doi: 10.1186/s40694-024-00179-0.
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.
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.
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.
指状青霉是一种真菌性植物病原菌，可导致采后柑橘类水果发生绿霉病。由于其经济重要性，许多研究致力于开发针对这种真菌的基因工程工具。此前已通过基于AMA1的自我复制质粒实现了CRISPR/Cas9技术的适应性改造，用于无标记基因编辑，但所得效率（10%）限制了其实际应用。在本研究中，我们旨在提高指状青霉中CRISPR/Cas9介导的基因编辑效率，以促进其实际应用。
在促进生长速度较慢的培养基中，通过在选择条件下进行额外的划线培养来延长培养时间，显著提高了指状青霉的基因编辑效率，最高可达54%-83%。为证明这一点，我们破坏了五个候选基因，这些基因是根据我们之前的高通量基因表达研究选择的，旨在阐明指状青霉对抗真菌蛋白PdAfpB的转录组反应。其中两个基因导致了明显的表型变化（PDIG_53730/pksP和PDIG_54100/arp2），并据此开始了方案优化。其他三个候选基因（PDIG_56860、PDIG_33760/rodA和PDIG_68680/dfg5）没有明显相关的表型，对其进行靶向操作以确认该方案的高效率。
通过改进选择方法，指状青霉的基因组编辑效率从10%显著提高到了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中CRISPR/Cas9基因编辑的效率，目前这些物种通过CRISPR/Cas9进行编辑的效率仍然较低。
