A Mad7 System for Genetic Engineering of Filamentous Fungi

Abstract

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.

Keywords: Aspergillus; CRISPR; Mad7; fungal strain engineering.

Figure 1

A Mad7 system for genetic engineering of fungi. (A) Schematic representations of Cas9 (left) and Mad7 (right) CRISPR nucleases. Scissile bonds in the target sequences are marked with orange triangles [24,25]. The positions of PAM sequences, as well as the orientation and the pairing of the sgRNA of Cas9 (fusion of tracrRNA and crRNA is indicated by the dotted line) and the gRNA of Mad7 (note that Mad7 has a natural single gRNA(=crRNA)) to the target template, are shown. (B) Mad7 vectors available for fungal genetic engineering. The vectors are shuttle vectors that can propagate in E. coli using ampicillin selection and in fungi using the AMA1 sequence and one of the selection markers indicated in the box. All vectors contain a mad7 gene and a USER cassette for insertion of the gRNA encoding gene by e.g., USER fusion of two PCR fragments, see Figure S1. Expression of the gRNA gene results in a composite transcript where the gRNA is released by the endogenous tRNA maturation machinery; see text for details.

Figure 2

Mad7-induced gene editing of the yA locus in A. nidulans. (A) Strategies for introducing a specific stop-codon/XbaI mutation (left) and an mRFP insertion (right) into yA. A DNA DSB is introduced in yA by the Mad7/yA-gRNA CRISPR nuclease. A single-stranded oligonucleotide is used as RT to direct introduction of stop-codon/XbaI mutation into yA by homologous recombination (HR). A PCR fragment is used as RT to direct insertion of mRFP into yA by HR. See main text for further details. (B) Mutation of yA changes the conidia color of transformants growing on solid medium from wild-type green to yellow. (C) To the left, transformation controls with empty Mad7-CRISPR plasmid (pDIV298) images in visible or in red fluorescent light (RFP); and with the yA-Mad7-CRISPR plasmid (pDIV711) in the absence of a RT. In the middle, introduction of the stop-codon/XbaI mutation into yA by co-transforming A. nidulans with pDIV711 and a single-stranded oligonucleotide (PR_DIV3197) serving as RT. RT mediated repair introduces an XbaI site and an amber stop codon. To the right, insertion of mRFP into yA by co-transforming A. nidulans with plasmid (pDIV711) and a mRFP-PCR fragment serving as RT.

Figure 3

Efficient Mad7-induced mutagenesis in A. niger. (A) Scheme illustrating the two-phase transformant recovery scheme. Protoplasts transformed with a Mad7-CRISPR plasmid are mixed with melted medium containing low stringency hygromycin concentration and plated as the first layer; see Materials and Methods for details. When media solidifies, plates are incubated at 30 °C favoring growth of A. niger, or 37 °C favoring Mad7 activity. After one day of incubation, overlay medium containing high-stringency hygromycin concentration is added to form the second layer, and plates are then transferred for further incubation at the temperature indicated. (B) Mad7-induced mutagenesis of A. niger albA at a specific position indicated by a red X (left) and phenotypic consequence of mutation of colonies, which changes conidia color from wild-type black to mutant albA white (right). (C) Experiment showing Mad7-induced mutagenesis of albA. Transformation of protoplasts using the empty Mad7-CRISPR plasmid (indicated as control) or with the albA Mad7-CRISPR vector in three independent trials (indicated as T1–T3). All experiments were plated at two different temperatures as indicated. For trials T1–T3, numbers of transformants with specific phenotypes are indicated below plates: B, Black; H, Heterokaryon (black and white); W, White.

Figure 4

Mad7-induced gene editing of the albA locus in A. niger. (A) Strategies for introducing a specific XbaI site/amber stop codon into albA (left) and an mRFP insertion (right) into albA locus. A DNA DSB is introduced in albA locus by Mad7 containing an albA specific gRNA. A single-stranded oligonucleotide is used as RT to direct introduction of the XbaI site/amber stop codon by HR. A PCR fragment is used as RT to direct insertion of mRFP into albA by HR. See main text for further details. (B) Mutation of albA changes the conidia color of transformants growing on solid medium from wild-type black to white. (C) To the left, transformation controls with empty Mad7-CRISPR plasmid (pDIV300) imaged in visible and red fluorescent light as indicated; and with the albA-Mad7-CRISPR plasmid (pDIV313) in the absence of a (RT). In the middle, introduction of a stop-codon mutation into albA using the albA Mad7-CRISPR plasmid (pDIV313) and an oligonucleotide (PR_DIV3196)-based RT. To the right, insertion of mRFP into albA using the albA-Mad7-CRISPR plasmid (pDIV313) and the PCR-based RT. Plates were imaged with visible and red fluorescent light as indicated.