Publication: Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277
Issued Date
2021-12-01
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ISSN
14726750
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2-s2.0-85101003042
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Mahidol University
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SCOPUS
Bibliographic Citation
BMC Biotechnology. Vol.21, No.1 (2021)
Suggested Citation
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. Vol.21, No.1 (2021). doi:10.1186/s12896-021-00669-8 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/75951
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Title
Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277
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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.
