Abstract
BackgroundCRISPR/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.ResultsWe 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.ConclusionsDirect 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.
Highlights
CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production
It was first found that the intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414
Expressed Cas9 led to unexpected offtarget genome editing The cas9 gene has been expressed constitutively or induced successfully in T. reesei, in both cases which can be used in successful genome editing in QM6a and RUT-C30 [7]
Summary
CRISPR/Cas has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. Trichoderma reesei is a well-known filamentous fungus workhorse for production of cellulase and other industrially important proteins [1]. Gene disruption is a critical technique for functional genomics study of T. reesei. Removal of major cellulase and protease genes is an essential step towards creating a platform strain for heterologous protein production [3]. A method is needed to efficiently disrupt the genes of interest in T. reesei. In T. reesei, gene disruption is routinely achieved by gene replacement through homology-based DNA recombination (HDR) [4]. One should note that the efficiency of HDR in T. reesei is notoriously low
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