Trichoderma Reesei using CRISPR

Lignocellulosic biomass, consisting mostly of cellulose, hemi-cellulose, and lignin, is the most abundant and renewable energy source on earth . Degradation of lignocellulosic biomass and continuation of the carbon cycle in nature is maintained mainly by microbial action, including different fungal species, such as Trichoderma, Aspergillus, and Penicillium. Microbes play important roles in nature. The biomass-degrading enzymes produced by these organisms also have applications in various fields of industry including food, fodder, paper, and textile industries. Trichoderma reesei, as one kind of fungus, is a well-known efficient producer of cellulase and hemi-cellulase, and is therefore widely employed by the enzyme industry for production of its own endogenous enzymes as well as production of heterogeneous proteins.Over the past 2 years, many studies have demonstrated that the CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations in genomesin a variety of organisms. Until now, there have been no reports on the CRISPR/Cas9 system or othergenome-editing approaches in filamentous fungi, even in the model organism Neurospora crassa, despite the successful application of this technique in yeast.

Trichoderma reesei (teleomorph Hypocrea jecorina) is a mesophilic soft-rot ascomycete fungi that is widely used in industry as a source of cellulases and hemicellulases for the hydrolysis of plant cell wall polysaccharides. Lignocellulosic biomass from agricultural crop residues, grasses, wood and municipal solid waste represents an abundant renewable resource that is becoming increasingly important as a future source of biofuels. Microbes are environmentally friendly livers on the earth. Although replacement of gasoline with cellulosic ethanol may substantially reduce greenhouse gases in the atmosphere and decrease global warming, the high cost of hydrolyzing biomass polysaccharides to fermentable sugars remains a major obstacle that must be overcome before cellulosic ethanol can be effectively commercialized. As the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, much cheaper sources of these enzymes are needed. Thus, genetic engineering techniques, gene knockout protocols and DNA-mediated transformation systems have improved industrial enzyme–producing T. reesei strains.

Efficient genome editing in Trichoderma reesei using the CRISPR/Cas9 system

Researchers demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

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

Researchers tested two gene disruption methods in the fungus 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.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.

A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei.

Researchers incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in one sort of fungus, T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media.

we developed an RNAi-mediated silencing system driven by the P tcu1 promoter which is highly responsive to the copper ions. The developed RNAi system could readily knock down/off the target gene in the absence of copper allowing the phenotypical characterization and could mimic the complementation of the deficient strain simply by including copper in the media to exclude the unwanted effect that may result from the random integration of the hpRNA cassette. The copper-responsive RNAi-mediated silencing system is applicable on different nutritional states and represents a powerful tool for characterizing target gene functions in T. reesei.

Reference

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