Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi

Abstract

Background: CRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan-chitin crosslinking enzymes involved in cell wall biosynthesis.

Results: Here, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9-sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9-sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members.

Conclusions: Our findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.

Keywords: Aspergillus niger; CRISPR/Cas9; Cell wall; Gene editing; Gene families; Knockouts; Marker free; Multiplexing; pyrG marker effects.

Fig. 1

Diagnostic PCR of crhA-G in the A. niger TLF39 and wild type (MA234.1) strains. ORFs removed for each crh gene in TLF39 show a downward band shift compared to MA234.1. a Exact band sizes are listed. b gDNA of TLF39 and MA234.1 was amplified with primer pairs for each crh gene (listed in Additional file 4: Table S1); PCR samples were loaded on 1% agarose gels

Fig. 2

Growth morphology of crh knockouts in MA234.1 (ΔkusA) obtained using CRISPR/Cas9. MA234.1, ΔcrhA, ΔcrhB, ΔcrhC, ΔcrhD, ΔcrhE, ΔcrhF, ΔcrhG, ΔcrhEF, ΔcrhDEF, ΔcrhABDEF, ΔcrhADEFG, ΔcrhABDEFG and ΔcrhABCDEFG on MM (a, b), MM + 400 µg/mL CFW (c, d) or MM + 800 µg/mL CR (e, f)

Fig. 3

Schematic representation of the pFC332_Pro¹-sgRNA plasmid construction. a Amplification of the two flanks that represent the Pro¹-sgRNA expression cassette: pTE1_for and pRC-target are used to amplify the Pro¹-tRNA promoter and target sequence, where pRC-target contains a variable 20 bp overhang (indicated by brown color) that represents the reverse complement target sequence of choice. In turn, pTarget and pTE1_rev are used to amplify the target-sgRNA-Pro¹-tRNA terminator flank. Here, pTarget contains a variable overhang that contains the target sequence of choice. Separate flanks are joined together through fusion PCR by pTE1_for and pTE1_rev, where the overhang sequence (=target) facilitates the homologous region between both flanks. b Addition of PacI sites to either end of the fusion construct (part of pTE1_for and pTE1_rev sequence) allows ligation of the fusion construct into pFC332. Diagnostic restriction analysis of the cloned plasmid ought to be done with Cfr42i (SacII) and shows a fragment of either 497 bp or 500 bp in addition to 1 kb and 14.3 kb fragments, for forward or reverse orientation, respectively

Fig. 4

Construction of marker free repair DNA fragment. Amplification of regions upstream (5′ flank) and downstream (3′ flank) of a gene of interest (GOI): Primer 1 and Primer 3 are used to amplify the 5′ flank, typically directly upstream from the start codon of the GOI. Primer 2 and Primer 4 are used to amplify the 3′ flank, just downstream of the ORF stop codon. Addition of 20 bp reverse complement sequence of Primer 3 to the 5′ end of Primer 2 ensures overlap between the 5′ and 3′ flanks necessary for fusion of the two flanks to construct the marker free repair DNA fragment. Upon introduction of the marker free repair DNA fragment to the fungal cell, repair of the double strand break (DSB) induced by a Cas9–sgRNA complex is possible by homology directed repair (HDR) at the site of the GOI