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Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277
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Document Title
Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277
Author
Abdulrachman D, Eurwilaichitr L, Champreda V, Chantasingh D, Pootanakit K
Name from Authors Collection
Affiliations
Mahidol University; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC); National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
BMC BIOTECHNOLOGY
Year
2021
Volume
21
Issue
2
Open Access
gold, Green Published
Publisher
BMC
DOI
10.1186/s12896-021-00669-8
Format
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.
Keyword
5-FOA | Aspergillus | Cpf1 | CRISPR | Filamentous fungi | FnCpf1 | Gene editing | pyrG
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
Ministry of Science and Technology (NSTDA), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand [P-18-51580, P-18-52106]; ASEAN Royal Golden Jubilee Scholarship (RGJ-ASEAN); Mahidol University Postgraduate scholarship; Thailand Graduate Institute of Science and Technology (TGIST), NSTDA
License
CC-BY
Rights
Authors
Publication Source
WOS