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 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.

Keywords: 5-FOA; Aspergillus; CRISPR/Cpf1; Filamentous fungi; FnCpf1; Gene editing; pyrG.

Fig. 1

Schematic of CRISPR/Cpf1 in a single-plasmid system for use in fungal genome engineering application. In this study, FnCpf1 and crRNA are expressed under A. nidulans TEF1 Pol II promoter and A. fumigatus U3 Pol III promoter, respectively, to edit genome of the wild-type strain of A. aculeatus TBRC 277

Fig. 2

Schematic of vector constructions for heterologous FnCpf1 expression and confocal microscopy assessment of the subcellular localization of the recombinant eGFP and Cpf1-EGFP fusion proteins in A. aculeatus TBRC 277. a The constructed vectors of (1) pCRISPR01-EGFP, expression of enhanced green fluorescence protein (EGFP), to test the tef1 promoter activity, (2) pCRISPR01-FnCpf1, for subsequent crRNA cassette insertion carrying a protospacer pyrG-targeted locus, (3) pCRISPR01-FnCpf1-EGFP, expression of Cpf1-EGFP, to detect Cpf1 fusion protein. b Subcelullar localization of FnCpf1 as EGFP-fused protein in A. aculeatus TBRC 277. Nuclei were visualized by DAPI staining (blue). Scale bar = 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein

Fig. 3

The complete sequence of pyrG gene of A. aculeatus TBRC 277 (GenBank accession number MN364695). The locations of the three selected protospacers are indicated by the red lines, with the corresponding 5′-TTTN-3′ PAM in blue (putative intron 158–238; 81 bp)

Fig. 4

An example of A. aculeatus TBRC 277 protoplasts transformed with CRISPR/Cpf1 plasmids. a pCRISPR01-FnCpf1-pyrGs (with crRNA specific targeting pyrG gene) on primary selective transformation medium containing 1.2 M sorbitol + 10 mM uri/ura + 50 μg/ml bleomycin, b a single colony selected from panel a, was then cultured on new minimal medium containing 10 mM uri/ura+ 50 μg/ml bleomycin, (c) secondary selection from panel b, re-cultured on minimal medium containing 10 mM uri/ura and 1.5 mg/ml 5-FOA. d pCRISPR01-FnCpf1 encoding only Cpf1 (without crRNA targeting pyrG), after growing on secondary selective medium containing 5-FOA, no colony was observed (control). Uri: uridine, Ura: uracil

Fig. 5

Indels of pyrG gene in A. aculeatus TBRC 277 genome transformed with CRISPR/Cpf1 plasmids with three different crRNAs. Red arrow indicates putative cleavage sites; red dashes, deleted bases; red bases, insertions or mutations