A tRNA-gRNA array-based CRISPR system for efficient and precise marker-free multiple gene editing of Trichoderma reesei to enhance cellobiose production

Cellobiose can be catalyzed by cellobiose phosphorylase to produce glucose-1-phosphate, which is then further synthesized into starch. This is a more energy efficient process compared to starch synthesis from glucose. The filamentous fungi Trichoderma reesei has the ability to secrete large amounts of cellulases to degrade cellulose to cellobiose, then continue to release glucose as the end product. In this study, we developed a highly efficient, marker-free and recyclable multigene editing system based on a T. reesei strain PXK1 with consecutively activated Xyrl A824V and deletion of ku70 gene with increased homologous recombination efficiency. The gene editing system which contained the CRISPR-Cas9 framework, the AMA1-based plasmid, and an intrinsic tRNA processing mechanism showed up to 100 %, 82 % and 67 % efficiency for editing one, two and three genes, respectively. Eleven single strains with single β-glucosidase (BG) deletion and then several strains with double and triple β-glucosidase deletions were obtained. The effect of BG deletions on cellobiose production were evaluated. The strain Δ cel3a showed a 128 % increase in cellobiose yield compared to strain PXK1, and the strain with cumulative deletion of two BG genes can efficiently produce 12 % more cellobiose than Δ cel3a . • Evaluated the effect of 11 individual β-glucosidase genes on cellobiose production. • Cas9 expression is the main limiting factor of the gene editing system. • tRNA-sgRNA-based system achieves 67 % efficiency in simultaneous triple-gene editing.