Dede Abdulrachman1, Lily Eurwilaichitr2, Verawat Champreda3, Duriya Chantasingh4, Kusol Pootanakit5. 1. Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand. 2. Thailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Khlong Luang District, Pathumthani, Thailand. 3. Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Khlong Luang District, Pathumthani, Thailand. 4. Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Khlong Luang District, Pathumthani, Thailand. duriya@biotec.or.th. 5. Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand. kusol.poo@mahidol.ac.th.
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.
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/Cpf1expression vector that targets pyrG gene in Aspergillus aculeatusTBRC 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. aculeatusTBRC 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.
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