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

2020 ◽  
Vol 42 (7) ◽  
pp. 1203-1210 ◽  
Author(s):  
Chuan Wu ◽  
Yumeng Chen ◽  
Yifei Qiu ◽  
Xiao Niu ◽  
Ningjian Zhu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
Simple Approach

scholarly journals Mappingin vivogenetic interactomics through Cpf1 crRNA array screening

2017 ◽  
Author(s):  
Ryan D. Chow ◽  
Guangchuan Wang ◽  
Adan Codina ◽  
Lupeng Ye ◽  
Sidi Chen
Keyword(s):  
Genome Editing ◽  
Biological Networks ◽  
Mammalian Species ◽  
Simple Approach ◽  
Genetic Interactions ◽  
Epigenetic Factors ◽  
Double Knockout ◽  
Mammalian Genomes ◽  
Single Effector

AbstractGenetic interactions lay the foundation of biological networks in virtually all organisms. Due to the complexity of mammalian genomes and cellular architectures, unbiased mapping of genetic interactionsin vivois challenging. Cpf1 is a single effector RNA-guided nuclease that enables multiplexed genome editing using crRNA arrays. Here we designed a Cpf1 crRNA array library targeting all pairwise permutations of the most significantly mutated nononcogenes, and performed double knockout screens in mice using a model of malignant transformation as well as a model of metastasis. CrRNA array sequencing revealed a quantitative landscape of all single and double knockouts. Enrichment, synergy and clonal analyses identified many unpredicted drivers and co-drivers of transformation and metastasis, with epigenetic factors as hubs of these highly connected networks. Our study demonstrates a powerful yet simple approach forin vivomapping of unbiased genetic interactomes in mammalian species at a phenotypic level.

scholarly journals In vivo synthesis of complex N -glycans by expression of human N -acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei

FEBS Letters ◽  
1999 ◽  
Vol 452 (3) ◽  
pp. 365-370 ◽  
Author(s):  
Marleen Maras ◽  
André De Bruyn ◽  
Wouter Vervecken ◽  
Jaana Uusitalo ◽  
Merja Penttilä ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
In Vivo Synthesis

scholarly journals Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system

2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Rui Liu ◽  
Ling Chen ◽  
Yanping Jiang ◽  
Zhihua Zhou ◽  
Gen Zou
Keyword(s):  
Genome Editing ◽  
Trichoderma Reesei ◽  
Filamentous Fungus

scholarly journals cAMP activates calcium signalling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yumeng Chen ◽  
Xingjia Fan ◽  
Xinqing Zhao ◽  
Yaling Shen ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Phospholipase C ◽  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
Calcium Signalling ◽  
Signal Transduction Pathway ◽  
Cellulase Production ◽  
Signalling Pathway ◽  
Dependent Manner ◽  
Transduction Pathway

Abstract Background The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. Results We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. Conclusions The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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