Editing Aspergillus terreus using the CRISPR-Cas9 system

Abstract

CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system to various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in Aspergillus terreus. In this study, we present CRISPR/Cas9 genome editing in A. terreus. To optimize the guide ribonucleic acid (gRNA) expression, we constructed a modified single-guide ribonucleic acid (sgRNA)/Cas9 expression plasmid. By co-transforming an sgRNA/Cas9 expression plasmid along with maker-free donor deoxyribonucleic acid (DNA), we precisely disrupted the lovB and lovR genes, respectively, and created targeted gene insertion (lovF gene) and iterative gene editing in A. terreus (lovF and lovR genes). Furthermore, co-delivering two sgRNA/Cas9 expression plasmids resulted in precise gene deletion (with donor DNA) in the ku70 and pyrG genes, respectively, and efficient removal of the DNA between the two gRNA targeting sites (no donor DNA) in the pyrG gene. Our results showed that the CRISPR/Cas9 system is a powerful tool for precise genome editing in A. terreus, and our approach provides a great potential for manipulating targeted genes and contributions to gene functional study of A. terreus.

Keywords: Aspergillus terreus; CRISPR-Cas9; genome-editing technology; marker-free donor DNA.

Graphical abstract

Figure 1.

The genome-editing system in A. terreus. Gene editing in A. terreus was performed using a modified pFC332 plasmid, and A. nidulans gpdA promoter of pFC332 plasmid was replaced by A. terreus gpdA promoter to express sgRNA. Circular and linear donor DNAs were applied to enhance the precise genome editing. The inner primers were used to detect the donor DNA, and the combined inner primers with outer primers were used to validate the gene editing.

Figure 2.

Generation of lovB deletion mutant. (a) Diagram showing lovB gene deletion. (b) The 281-bp band represents the colony PCR product amplified from the donor plasmid of lovB gene. (c) Genome PCR analysis revealed that lovB gene was deleted, resulting in an expected band of 695 bp. Lane 1: wild-type (Wt) strain, lane 2: edited strain and lane M: 100-bp DNA marker.

Figure 3.

Isolation of fully edited strains by streaking method. Before streaking, bands of both edited and unedited strains can be seen in transformants. After applying for streaking, only the edited band was observed in the fully edited strain. (a) Diagram representing lovR gene deletion. (b) Generation of homokaryotic mutants of the lovR gene. Wt: wild-type and M: 100-bp DNA marker.

Figure 4.

Isolation of fully edited strains by streaking method. (a) Diagram showing lovF gene insertion. (b) Generation of DNA-inserted homokaryotic strain. Wt: wild-type and M: 100-bp DNA marker.

Figure 5.

Iterative genome editing in A. terreus. After the plasmid removal process, the plasmid-free lovF mutant was used to apply the deletion of the lovR gene. The target mutations were detected by genome PCR using the corresponding primers. From left to right: the 414-bp band represented the unedited genome, and the 1014-bp band represented the inserted lovF. For the second genome editing, the 937-bp band represented the unedited genome, and the 724-bp band represented lovR deletion. Wt: wild-type, Mut: edited mutant and M: 100-bp DNA marker.

Figure 6.

Generation of gene deletion with two sgRNAs. (a) Diagram representing ku70 gene deletion. (b) An 833-bp band corresponding to the size of the deleted ku70 was observed on the agarose gel. Wt: wild-type and M: 100-bp DNA marker.

Figure 7.

Generation of gene deletion with two sgRNAs. (a) Diagram showing pyrG gene deletion. (b) Genome PCR analysis showed that pyrG gene was deleted, resulting in pyrG^- (homokaryotic deletion) and pyrG^± mutants (heterokaryotic deletion). (c) Wild-type and mutant strains were checked on PDA medium, PDA medium with uracil and PDA medium containing uracil and 5-FOA. Wt: wild-type and M: 100-bp DNA marker.

Figure 8.

Targeted deletion of pyrG by NHEJ. (a) Diagram representing pyrG gene deletion via NHEJ. (b) pyrG mutations were determined by PCR. Sequence analysis of pyrG deletion, the sgRNA sequence was shown in green and the PAM region was highlighted in dark pink. Break sites were indicated by yellow arrowheads. M indicates100-bp DNA marker.