Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277
Abstract Background CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged a...
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| Format: | Article |
| Language: | English |
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BMC
2021-02-01
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| Series: | BMC Biotechnology |
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| Online Access: | https://doi.org/10.1186/s12896-021-00669-8 |
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| author | Dede Abdulrachman Lily Eurwilaichitr Verawat Champreda Duriya Chantasingh Kusol Pootanakit |
| author_facet | Dede Abdulrachman Lily Eurwilaichitr Verawat Champreda Duriya Chantasingh Kusol Pootanakit |
| author_sort | Dede Abdulrachman |
| collection | DOAJ |
| description | Abstract Background CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi. Results In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci. Conclusions The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi. |
| first_indexed | 2024-12-17T04:50:32Z |
| format | Article |
| id | doaj.art-067c59e17545427f81583a61d047bb46 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-17T04:50:32Z |
| publishDate | 2021-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-067c59e17545427f81583a61d047bb462022-12-21T22:02:54ZengBMCBMC Biotechnology1472-67502021-02-0121111310.1186/s12896-021-00669-8Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277Dede Abdulrachman0Lily Eurwilaichitr1Verawat Champreda2Duriya Chantasingh3Kusol Pootanakit4Institute of Molecular Biosciences, Mahidol UniversityThailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science ParkEnzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science ParkEnzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science ParkInstitute of Molecular Biosciences, Mahidol UniversityAbstract Background CRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi. Results In this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci. Conclusions The use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.https://doi.org/10.1186/s12896-021-00669-8CRISPR/Cpf1pyrG5-FOAFnCpf1Gene editingFilamentous fungi |
| spellingShingle | Dede Abdulrachman Lily Eurwilaichitr Verawat Champreda Duriya Chantasingh Kusol Pootanakit Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 BMC Biotechnology CRISPR/Cpf1 pyrG 5-FOA FnCpf1 Gene editing Filamentous fungi |
| title | Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 |
| title_full | Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 |
| title_fullStr | Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 |
| title_full_unstemmed | Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 |
| title_short | Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277 |
| title_sort | development of a crispr cpf1 system for targeted gene disruption in aspergillus aculeatus tbrc 277 |
| topic | CRISPR/Cpf1 pyrG 5-FOA FnCpf1 Gene editing Filamentous fungi |
| url | https://doi.org/10.1186/s12896-021-00669-8 |
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