Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

Area of Science:

• Molecular Biology
• Biotechnology
• Mycology

Background:

• CRISPR/Cas9 technology has broad applications in biological species, including Trichoderma reesei, a key fungus for cellulase production.
• Heterologous Cas9 expression in host cells can be challenging and time-consuming.

Purpose of the Study:

• To evaluate two CRISPR/Cas9 gene disruption methods in Trichoderma reesei.
• To establish an efficient genome editing approach for T. reesei.

Main Methods:

• Testing intracellular Cas9 expression for gene disruption, observing off-target effects.
• Developing an in vitro assembly of Cas9 and guide RNA (gRNA), followed by ribonucleoprotein complex transformation.
• Utilizing a plasmid with a pyr4 marker gene for transformation into T. reesei TU-6.
• Targeting the cbh1 gene using gRNA and assessing gene disruption in transformants.

Main Results:

• Intracellular Cas9 expression resulted in unintended insertions near the ura5 gene in T. reesei QM9414.
• The in vitro assembled Cas9/gRNA complex successfully disrupted the cbh1 gene in T. reesei TU-6.
• Eight out of 27 transformants showed cbh1 gene disruption, confirmed by loss of CBH1 expression.
• Large DNA fragments were inserted at the disrupted cbh1 locus.

Conclusions:

• Direct transformation of the Cas9/gRNA complex is an efficient and rapid method for gene disruption in T. reesei.
• This approach has significant potential for applications in fungal strain improvement and functional genomics studies.