An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production
Abstract Background The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-02-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13068-024-02468-7 |
| _version_ | 1797275463024902144 |
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| author | Jiaxin Zhang Kehang Li Yu Sun Cheng Yao Weifeng Liu Hong Liu Yaohua Zhong |
| author_facet | Jiaxin Zhang Kehang Li Yu Sun Cheng Yao Weifeng Liu Hong Liu Yaohua Zhong |
| author_sort | Jiaxin Zhang |
| collection | DOAJ |
| description | Abstract Background The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Results Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA−sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA−sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77 U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. Conclusions The tRNA−gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology. |
| first_indexed | 2024-03-07T15:14:43Z |
| format | Article |
| id | doaj.art-1bd40b31d8994663b908229ac0277b72 |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-03-07T15:14:43Z |
| publishDate | 2024-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-1bd40b31d8994663b908229ac0277b722024-03-05T17:57:50ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542024-02-0117111810.1186/s13068-024-02468-7An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme productionJiaxin Zhang0Kehang Li1Yu Sun2Cheng Yao3Weifeng Liu4Hong Liu5Yaohua Zhong6State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityAbstract Background The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Results Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA−sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA−sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77 U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. Conclusions The tRNA−gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.https://doi.org/10.1186/s13068-024-02468-7CRISPR/Cas9Trichoderma reeseiMultigene editingGlucose oxidaseLignocellulose-degrading enzymesMultiple sgRNA processing |
| spellingShingle | Jiaxin Zhang Kehang Li Yu Sun Cheng Yao Weifeng Liu Hong Liu Yaohua Zhong An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production Biotechnology for Biofuels and Bioproducts CRISPR/Cas9 Trichoderma reesei Multigene editing Glucose oxidase Lignocellulose-degrading enzymes Multiple sgRNA processing |
| title | An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production |
| title_full | An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production |
| title_fullStr | An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production |
| title_full_unstemmed | An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production |
| title_short | An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production |
| title_sort | efficient crispr cas9 genome editing system based on a multiple sgrna processing platform in trichoderma reesei for strain improvement and enzyme production |
| topic | CRISPR/Cas9 Trichoderma reesei Multigene editing Glucose oxidase Lignocellulose-degrading enzymes Multiple sgRNA processing |
| url | https://doi.org/10.1186/s13068-024-02468-7 |
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