Trichoderma harzianum marker-free strain construction based on efficient CRISPR/Cas9 recyclable system: A helpful tool for the study of biological control agents

This article reports the first successful marker-free genetic engineering of Trichoderma harzianum, a widely studied biocontrol agent with great potential for use in sustainable agriculture. T. harzianum has been shown to control plant diseases, promote plant growth, and improve plant tolerance to abiotic stress. A specific CRISPR/Cas9 gene-editing system, based in self-replicative AMA1-plasmid, was used to generate T. harzianum mutant strains, targeting the albA (pks4) and ku70 genes, involved in melanin synthesis and non-homologous end joining DNA repair pathway, respectively. The resulting albA-deficient strain displayed a loss of pigmentation in their conidia, confirming phenotypically and genotypically the efficacy of the CRISPR/Cas9 system in generating targeted gene knockouts in T. harzianum. A marker-free Δku70 strain was also constructed, with the maintenance of its original fitness, providing a helpful tool for precise genetic engineering in T. harzianum, due to NHEJ-deficiency, able to be used in in vitro and in vivo (in planta) studies. The study highlights the potential of using CRISPR/Cas9 technology to facilitate the study of the molecular processes involved in the biocontrol and plant growth-promoting properties of T. harzianum.