Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277

• Published in: BMC biotechnology Vol. 21; no. 1; p. 15
• Main Authors: Abdulrachman, Dede, Eurwilaichitr, Lily, Champreda, Verawat, Chantasingh, Duriya, Pootanakit, Kusol
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 11.02.2021; BioMed Central; BMC
• Subjects: 5-FOA; Aspergillus; Aspergillus - genetics; Aspergillus aculeatus; Bacterial Proteins - genetics; Biotechnology; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; CRISPR/Cpf1; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Endonucleases - genetics; Enzymes; Feasibility studies; Filamentous fungi; FnCpf1; Francisella; Fungi; Gene disruption; Gene editing; Gene Editing - methods; Gene Expression Regulation, Fungal; gene targeting; Genes; Genetic aspects; Genetic engineering; genetic vectors; Genome editing; Genomes; Genomics; Homology; Localization; Methods; Microbial genetic engineering; Mutagenesis; Non-homologous end joining; Nuclease; Physiological aspects; Plasmids; pyrG; PyrG gene; Ribonucleic acid; RNA; RNA polymerase; RNA, Guide, CRISPR-Cas Systems; synthetic biology; Thailand; 5-FOA; CRISPR/Cpf1; Aspergillus; FnCpf1; Filamentous fungi; Gene editing; pyrG
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/s12896-021-00669-8

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. 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. 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.