CRISPR/Cas9 para obtenção de Trichoderma harzianum com marcas de seleção

Abstract

Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species and has not yet been reported. This work registers obtaining of mutants with selection marks in this species using the CRISPR/Cas9 system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biobalistics, and the sequencing approach proved mutants deletions and insertions at pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid; besides the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, it was possible to demonstrate target disruption of pyr4 gene in T. harzianum using the CRISPR/Cas9 system and also that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report for this technique in this species of biocontrol and, the mutants expressing Cas9, represent biotechnological potential for generation of useful technologies in agricultural biotechnology.