A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription

Wu, Chuan; Chen, Yumeng; Qiu, Yifei; Niu, Xiao Da; Zhu, Ningjian; Chen, Jiehui; Yao, Huilu; Wang, Wei; Ma, Yushu

doi:10.1007/s10529-020-02887-0

Cited by 19 publications

(11 citation statements)

References 28 publications

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“…The use of this method has been reported with respect to gene disruption in T. atroviride (Zeilinger, 2004 ) and was subsequently successfully developed for the transformation of T. reesei using hph (encoding hygromycin B phosphotransferase) as the selectable marker gene (Zhong et al, 2007 ). Furthermore, in 2019, a modified ATMT method using two different A. tumefaciens strains was reported that could be used to simultaneously introduce two plasmids in a single step (Wu, Chen, Huang, et al, 2020 ; Wu, Chen, Qiu, et al, 2020 ). Although AMAT is another common transformation method, it resulted in a lower efficiency of DNA integration and less stable transformants when the ATMT and PMT methods were compared in four different Trichoderma species (Cardoza et al, 2006 ).…”

Section: Transformation Methodsmentioning

confidence: 99%

“…Most applications of the CRISPR‐Cas system also have direct utility and relevance with respect to Trichoderm a, for which CRISPR‐Cas can be applied to augment and/or enhance pre‐existing genetic engineering platforms (Liu et al, 2015). To date, researchers have constructed CRISPR‐Cas tools based on diversification strategies and have applied them to functional gene identification, strain modification, and other fields in T. reesei (Bodie et al, 2021; Chai et al, 2022; de Souza Maia Filho et al, 2017; Hao & Su, 2019; Li, Lin et al, 2021; Li, Liu et al, 2021; Liu et al, 2015; Rantasalo et al, 2019; Wu, Chen, Huang, et al, 2020; Wu, Chen, Qiu, et al, 2020; Zou, Bao et al, 2021; Zou, Li et al, 2021; Zou, Xiao et al, 2021). Nevertheless, some Trichoderma species, including T. harzianum (Vieira et al, 2021), T. atroviride (Primerano, 2021), and the unidentified species Trichoderma sp.…”

Section: Introductionmentioning

confidence: 99%

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Use of CRISPR‐Cas tools to engineer Trichoderma species

Wang

Chen

et al. 2022

Microbial Biotechnology

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Given their lignocellulose degradability and biocontrol activities, fungi of the ubiquitously distributed genus Trichoderma have multiple industrial and agricultural applications. Genetic manipulation plays a valuable role in tailoring novel engineered strains with enhanced target traits. Nevertheless, as applied to fungi, the classic tools of genetic manipulation tend to be time‐consuming and tedious. However, the recent development of the CRISPR‐Cas system for gene editing has enabled researchers to achieve genome‐wide gene disruptions, gene replacements, and precise editing, and this technology has emerged as a primary focus for novel developments in engineered strains of Trichoderma. Here, we provide a brief overview of the traditional approaches to genetic manipulation, the different strategies employed in establishing CRSIPR‐Cas systems, the utilization of these systems to develop engineered strains of Trichoderma for desired applications, and the future trends in biotechnology.

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Section: Transformation Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Use of CRISPR‐Cas tools to engineer Trichoderma species

Wang

Chen

et al. 2022

Microbial Biotechnology

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“…Later on, the 5S rRNA promotor of A. niger was suggested for expression of the guide RNA, which enabled gene deletion using a donor DNA carrying only a 40 bp homology sequence and no selectable marker gene (Wang et al, 2021). Similarly, the promotors of two RNA polymerase III U6 snRNA genes were confirmed to be suitable for gRNA expression in T. reesei (Wu et al, 2020). Although the method of genome editing has become quite popular for modifications in fungi, the method via Cas9-CRISPR gRNA ribonucleoprotein complexes assembled in vitro is relatively low.…”

Section: Crispradaptation To Trichoderma and Optimizationmentioning

confidence: 99%

Trichoderma – genomes and genomics as treasure troves for research towards biology, biotechnology and agriculture

Schalamun

Schmoll

2022

Front. Fungal Biol.

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The genus Trichoderma is among the best studied groups of filamentous fungi, largely because of its high relevance in applications from agriculture to enzyme biosynthesis to biofuel production. However, the physiological competences of these fungi, that led to these beneficial applications are intriguing also from a scientific and ecological point of view. This review therefore summarizes recent developments in studies of fungal genomes, updates on previously started genome annotation efforts and novel discoveries as well as efforts towards bioprospecting for enzymes and bioactive compounds such as cellulases, enzymes degrading xenobiotics and metabolites with potential pharmaceutical value. Thereby insights are provided into genomes, mitochondrial genomes and genomes of mycoviruses of Trichoderma strains relevant for enzyme production, biocontrol and mycoremediation. In several cases, production of bioactive compounds could be associated with responsible genes or clusters and bioremediation capabilities could be supported or predicted using genome information. Insights into evolution of the genus Trichoderma revealed large scale horizontal gene transfer, predominantly of CAZyme genes, but also secondary metabolite clusters. Investigation of sexual development showed that Trichoderma species are competent of repeat induced point mutation (RIP) and in some cases, segmental aneuploidy was observed. Some random mutants finally gave away their crucial mutations like T. reesei QM9978 and QM9136 and the fertility defect of QM6a was traced back to its gene defect. The Trichoderma core genome was narrowed down to 7000 genes and gene clustering was investigated in the genomes of multiple species. Finally, recent developments in application of CRISPR/Cas9 in Trichoderma, cloning and expression strategies for the workhorse T. reesei as well as the use genome mining tools for bioprospecting Trichoderma are highlighted. The intriguing new findings on evolution, genomics and physiology highlight emerging trends and illustrate worthwhile perspectives in diverse fields of research with Trichoderma.

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“…Zou et al demonstrated that very short flanks (20 bp) could be used for gene deletions in combination with the Cas9 ribonucleoprotein [ 53 ]. Two other groups suggested using RNA polymerase III promoters for in vivo transcription of the sgRNA [ 56 , 57 ], in order to circumvent possible problems with in vitro transcribed sgRNA uptake and stability. In these studies, a plasmid containing an sgRNA expression cassette under the control of an RNA Pol III promoter was either transformed into an Cas9-expressing strain [ 56 ] or co-transformed together, with another plasmid bearing the cas9 gene [ 57 ].…”

Section: Application Of the Crispr/cas9 System For Genome Editingmentioning

confidence: 99%

“…Two other groups suggested using RNA polymerase III promoters for in vivo transcription of the sgRNA [ 56 , 57 ], in order to circumvent possible problems with in vitro transcribed sgRNA uptake and stability. In these studies, a plasmid containing an sgRNA expression cassette under the control of an RNA Pol III promoter was either transformed into an Cas9-expressing strain [ 56 ] or co-transformed together, with another plasmid bearing the cas9 gene [ 57 ]. Regardless of the used method, the CRISPR/Cas9 system promises to enhance the genome editing capabilities in Trichoderma spp.…”

Section: Application Of the Crispr/cas9 System For Genome Editingmentioning

confidence: 99%

An overview on current molecular tools for heterologous gene expression in Trichoderma

Tomico-Cuenca

Mach

Mach-Aigner

et al. 2021

Fungal Biol Biotechnol

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Fungi of the genus Trichoderma are routinely used as biocontrol agents and for the production of industrial enzymes. Trichoderma spp. are interesting hosts for heterologous gene expression because their saprotrophic and mycoparasitic lifestyles enable them to thrive on a large number of nutrient sources and some members of this genus are generally recognized as safe (GRAS status). In this review, we summarize and discuss several aspects involved in heterologous gene expression in Trichoderma, including transformation methods, genome editing strategies, native and synthetic expression systems and implications of protein secretion. This review focuses on the industrial workhorse Trichoderma reesei because this fungus is the best-studied member of this genus for protein expression and secretion. However, the discussed strategies and tools can be expected to be transferable to other Trichoderma species.

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A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription

Cited by 19 publications

References 28 publications

Use of CRISPR‐Cas tools to engineer Trichoderma species

Use of CRISPR‐Cas tools to engineer Trichoderma species

Trichoderma – genomes and genomics as treasure troves for research towards biology, biotechnology and agriculture

An overview on current molecular tools for heterologous gene expression in Trichoderma

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