ORIGINAL RESEARCH article
Front. Genome Ed.
Sec. Genome Editing Tools and Mechanisms
Volume 7 - 2025 | doi: 10.3389/fgeed.2025.1623963
Simply cut out -Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion in Trichoderma atroviride
Provisionally accepted
Mario Gründlinger
Chiara EllensohnLeo Drechsel
Ulrike SchreinerSiebe Pierson
Clara Baldin
Susanne Zeilinger*- Department of Microbiology, University of Innsbruck, Innsbruck, Austria
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Trichoderma atroviride 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-pks4 (spore pigment production), pyr4 (pyrimidine biosynthesis), and pex5 (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 (hph), 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 T. atroviride, offering a fast and reliable approach for reverse genetics in this agriculturally important fungus.
Keywords: Trichoderma atroviride, CRISPR/Cas9, telomeric vector, Transient resistance, marker-free
Received: 06 May 2025; Accepted: 18 Jun 2025.
Copyright: © 2025 Gründlinger, Ellensohn, Drechsel, Schreiner, Pierson, Baldin and Zeilinger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Susanne Zeilinger, Department of Microbiology, University of Innsbruck, Innsbruck, Austria
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