Gründlinger Mario, Ellensohn Chiara, Drechsel Leo, Schreiner Ulrike, Pierson Siebe, Baldin Clara, Zeilinger Susanne
Department of Microbiology, University of Innsbruck, Innsbruck, Austria.
Front Genome Ed. 2025 Jul 2;7:1623963. doi: 10.3389/fgeed.2025.1623963. eCollection 2025.
is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. However, genetic manipulation in this organism remains challenging due to the limited availability of versatile and efficient molecular tools. Here, we present a CRISPR/Cas9-based method for targeted gene manipulation using ribonucleoprotein (RNP) complexes combined with a transiently stable telomere vector. We successfully inactivated three genes- (spore pigment production), (pyrimidine biosynthesis), and (peroxisomal matrix protein import receptor)-to demonstrate the system's utility. Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene inactivations in our case were achieved via non-homologous end joining (NHEJ), by co-transforming the transiently stable telomere vector carrying the hygromycin-resistance gene (), which was rapidly lost under non-selective conditions. This strategy enables marker-free genetic manipulation, supports vector recycling, and simplifies successive transformations. Overall, our method expands the genetic toolbox for , offering a fast and reliable approach for reverse genetics in this agriculturally important fungus.
是一种广为人知的菌寄生真菌，广泛用于真菌植物病原体的生物防治。然而，由于通用且高效的分子工具有限，对该生物体进行基因操作仍然具有挑战性。在此，我们提出了一种基于CRISPR/Cas9的方法，使用核糖核蛋白（RNP）复合物结合瞬时稳定的端粒载体进行靶向基因操作。我们成功地使三个基因失活——（孢子色素产生）、（嘧啶生物合成）和（过氧化物酶体基质蛋白导入受体）——以证明该系统的实用性。虽然Cas9诱导的双链断裂可以通过同源定向修复（HDR）利用供体模板进行修复，但在我们的案例中，最有效的基因失活是通过非同源末端连接（NHEJ）实现的，通过共转化携带潮霉素抗性基因（）的瞬时稳定端粒载体，该载体在非选择性条件下会迅速丢失。这种策略能够实现无标记基因操作，支持载体循环利用，并简化连续转化。总体而言，我们的方法扩展了的基因工具箱，为这种在农业上具有重要意义的真菌的反向遗传学提供了一种快速可靠的方法。
