Abstract
BackgroundCRISPR/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.ResultsHere, 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.ConclusionsOur 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.
Highlights
The fungal cell wall is comprised of a series of different polymeric sugars, such as alpha-glucans, beta-glucans, chitin, galactomannans and mannoproteins
CRISPR/Cas9 plasmid design and proof of functionality Our CRISPR/Cas9 procedure combines Cas9 expression driven by the constitutive Aspergillus nidulans tef1promoter from an autonomously replicating plasmid with expression of the single guide RNA (sgRNA) driven by the tRNAPro1 RNAIII polymerase promoter [20]
To show functionality of the CRISPR/Cas9 system, we chose to target the brnA gene (NRRL3_01040; An14g05370) of A. niger which is homologous to Aspergillus fumigatus abr1 and encodes a multi copper oxidase that is involved in the synthesis of melanin [26, 27]
Summary
The fungal cell wall is comprised of a series of different polymeric sugars, such as alpha-glucans, beta-glucans, chitin (poly-1,4-linked N-acetyl-glucosamine), galactomannans and mannoproteins. Current methods to create multiple gene deletions in A. niger include the use of the pyrG or amdS-based transformation system combined with the subsequent recycling of pyrG or amdS via counter selection approach using 5-fluoroorotic acid (5-FOA) or 5-fluoro-acetamide (5-FAA) [11], respectively Another approach is the use of multiple auxotrophic strains, but limits one to a total of four separate selection markers in A. niger (pyrG, nicB, argB, adeA) [12]. CRISPR/Cas 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
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