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Background  CRISPR/Cas9 has wide application potentials in a variety of biological species including  Trichoderma reesei , a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome.    Results  We tested two gene disruption methods in  T. reesei  using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in  T. reesei  QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted  ura5 . An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the  pyr4  marker gene into  T. reesei  TU-6. When the gRNA targeting  cbh1  was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful  cbh1  disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted  cbh1  locus.    Conclusions  Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in  T. reesei  and may find wide applications in strain improvement and functional genomics study.    Electronic supplementary material  The online version of this article (10.1186/s12896-018-0498-y) contains supplementary material, which is available to authorized users.

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