Background: CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma\nreesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the\nhost cell could be time-consuming and sometimes even troublesome.\nResults: We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly\nexpressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at\n70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling\nCas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing\nthe pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven\ntransformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through\ngenome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of\nthese nucleotides, were inserted in the disrupted cbh1 locus.\nConclusions: Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei\nand may find wide applications in strain improvement and functional genomics study.
Loading....