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Letter
FUNCTIONAL RECONSTITUTION OF A FUNGAL NATURAL PRODUCT GENE CLUSTER BY ADVANCED GENOME EDITING Jakob Weber, Vito Valiante, Christina Spuur Nødvig, Derek Joseph Mattern, Rebecca A. Slotkowski, Uffe H. Mortensen, and Axel A. Brakhage ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00203 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 10, 2016
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FUNCTIONAL RECONSTITUTION OF A FUNGAL NATURAL PRODUCT
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GENE CLUSTER BY ADVANCED GENOME EDITING
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Jakob Weber [1,2], Vito Valiante [3], Christina S. Nødvig [4], Derek J. Mattern [1,2], Rebecca A.
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Slotkowski [1], Uffe H. Mortensen [4] and Axel A. Brakhage [1,2] *
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[1] Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and
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Infection Biology – Hans Knöll Institute (HKI), Jena, Germany
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[2] Institute of Microbiology, Friedrich Schiller University Jena, Germany
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[3] Leibniz Research Group – Biobricks of Microbial Natural Product Syntheses, Leibniz Institute for Natural
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Product Research and Infection Biology (HKI), Jena, Germany
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[4] Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology,
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Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark
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* Corresponding author:
[email protected] 13
Table of Contents Graphic
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Abstract
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Filamentous fungi produce varieties of natural products even in a strain dependent manner.
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However, the genetic basis of chemical speciation between strains is still widely unknown. One
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example is trypacidin, a natural product of the opportunistic human pathogen Aspergillus
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fumigatus, which is not produced amongst different isolates. Combining computational analysis
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with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase
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of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain.
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Thus, we present a CRISPR/Cas9-based tool for advanced molecular genetic studies in
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filamentous fungi, exploiting selectable markers separated from the edited locus.
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Keywords: CRISPR/Cas9, gene-editing, Aspergillus fumigatus, trypacidin, split-marker
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The human pathogen Aspergillus fumigatus has been shown to produce a plenitude of natural
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products (NPs) including some that have been linked to pathogenicity.1 Until now, the rise of
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genomics and transcriptomics has accelerated the identification of gene clusters and the
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elucidation of biosynthetic pathways for NPs. Furthermore, sequence data revealed a hidden
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treasure of putative NP clusters of so far unknown corresponding products.2,3 To overcome the
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obstacle of activating these silent gene clusters, various techniques for discovering new NPs in
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the laboratory have been developed. These techniques can range from activation by
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overexpressing pathway-specific transcription factors or global regulators to the simulation of
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environmental stimuli.4–6 Nevertheless, the elucidation of a new NP is dependent on the
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functionality of all enzymes involved in its biosynthesis. Mutations in genes such as transcription
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factors, polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), or tailoring
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enzymes could hinder the production of a NP.
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With the availability of an increasing amount of genomic data, the in silico analysis of putative
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pathway genes supports the identification of possible mutations. This has been shown in the
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investigation of fumitremorgin biosynthesis in the A. fumigatus strain Af293. While various
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strains of A. fumigatus are able to produce different fumitremorgin derivatives, it was
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demonstrated that a single point mutation in the cluster gene ftmD, coding for an O-
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methyltransferase, led to the abolishment of further fumitremorgin products in Af293.7 This study
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illustrated, that chemical specificity of different strains can be dependent on the functionality of a
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single gene involved in the respective biosynthetic pathway.
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Furthermore, in a study by Frisvad and colleagues (2009), 40 A. fumigatus strains of different
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origins were analyzed, showing that variations in the occurrence of many NPs were present
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among the different strains tested.8 In particular, trypacidin was detected in 30 of the analyzed
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strains, including the clinical isolate and common lab strain Af293. At the same time, the
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remaining 10, including a second clinical isolate and lab strain CEA10, lacked its production.8
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Interestingly, trypacidin is a conidium-borne NP, which has been known for decades and was
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originally shown to be antiprotozoal as well as toxic to human lung cells.9,10 Additionally, its
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potential role as a virulence determinant was described by showing how it can influence the
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phagocytosis of different cells such as murine alveolar macrophages and the amoeba
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Dictyostelium discoideum.11 Since NPs of the opportunistic human pathogen A. fumigatus could
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potentially be involved in infection, it is of great interest to analyze genetic differences in 3
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production of NPs. In the case of trypacidin, the recent identification of the trypacidin
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biosynthetic gene cluster11,12 has set the stage for this type of analysis.
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The trypacidin biosynthetic gene cluster was predicted to be composed of 13 genes. Nucleotide
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alignment of the available A. fumigatus genomes showed nonconservative single nucleotide
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polymorphisms (SNPs) in different strains. Moreover, in the genome of strain CEA10 a single
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nucleotide insertion in the PKS-coding gene tynC/tpcC was predicted, that potentially resulted in
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a frameshift and appearance of a premature stop codon.12
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By reverse transcription polymerase chain reaction (RT-PCR) we first evaluated that the mRNA
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of tynC in the A. fumigatus strains Af293 as well as CEA10 and its descendant strain CEA17
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∆akuBKU80 pyrG+ (∆akuBKU80) (see also Table S1) was produced (Figure S1). Therefore, lack of
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expression in strain CEA10 appeared not to be the reason for lack of trypacidin production.
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To further analyze whether any mutation was present in the genomic tynC sequence, the tynC
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genes from the three mentioned strains were sequenced in the area of the reported single
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nucleotide insertion. Alignment of the sequenced fragments confirmed the predicted frameshift in
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the strains CEA10 and ∆akuBKU80, along with no other nucleotide variations. Since the frameshift
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is assumed to cause a premature stop codon, this would lead to the loss of the predicted acyl
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carrier protein (ACP) and product template (PT) domains, necessary for the catalytic function of
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the PKS (Figure 1A). Nevertheless, it is worth noting that standard prediction programs (e.g.
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AUGUSTUS13) could not detect this modification and thus assigned the predicted gene as
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functional. Instead, an additional intron in the tynC gene of CEA10 at the site of the frameshift
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was predicted. Thereby, the mutation was regarded as silent in the deduced protein (Figure S2).
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Moreover, further known SNPs in the gene could also have an influence on the function of the
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enzyme.
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To analyze the influence of the single nucleotide insertion on the function of TynC in CEA10, we
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first followed a conventional strategy by replacing the putative dysfunctional allele of tynC with
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the functional tynC gene from the Af293 strain. For this purpose, tynC was initially deleted in
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∆akuBKU80. The resulting deletion mutant was then complemented with a construct derived from
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plasmid pJW tynC-ptrA comp(lementation; see also 11). This two-step procedure was necessary to
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ensure a successful integration of the complete gene. Deletion mutants of tynC as well as strains
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harboring the Af293 tynC gene were verified by Southern blot analyses (Figure S3). RT-PCR 4
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demonstrated the expression of tynC in the complemented strain (Figure S1). LC-MS analysis of
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stationary cultures of the latter strains revealed the production of trypacidin (Figure 2 and Figure
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S4) showing that the Af293 tynC gene complemented the lack of trypacidin production in
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akuBKU80. This finding further suggested that the single nucleotide mutation resulted in a
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dysfunctional gene. Nevertheless, at this stage we could not exclude a possible role of further
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SNPs in the tynC of CEA10.
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As shown, editing single nucleotides by gene replacement is time-consuming and encompasses
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two or more steps. First, the deletion of the target gene has to be accomplished followed by a
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subsequent complementation of the respective knock-out mutant with a functional version of the
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gene. In case of tynC, the target gene was directly cloned from a producing strain (Af293) into a
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plasmid without any changes. However, in the case that functional genes are not available,
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nonfunctional genes need to be isolated and cloned in an appropriate vector. Then, editing has to
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be performed by site-directed mutagenesis, before complementing the mutant strain.
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Furthermore, it is not clear whether changes in promoter or terminator as well as the position of
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the selection marker upstream or downstream of the transferred gene could potentially interfere
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with expression of the latter. To avoid this, the Cre/loxP or other comparable systems could be
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used to subsequently remove the marker again.14 Nevertheless, these systems involve additional
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working steps to excise the marker cassette, which leaves scars in the genome with unknown
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potential effects.
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An alternative is provided by the latest development in genome editing, the clustered, regularly
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interspaced, short palindromic repeat (CRISPR) technology, which has expanded the toolbox of
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precise molecular scissors such as zinc finger nucleases (ZFN) and TALENs (transcription
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activator-like effector nucleases).15 Thereby, a CRISPR-associated endonuclease Cas9 originally
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derived from Streptococcus pyogenes combined with a synthetic guide RNA (gRNA) operate as a
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functional unit. The gRNA provides a 20 bp DNA recognition site (spacer) for Cas9. The protein-
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RNA complex binds to the respective target site of the spacer (protospacer) in the genome and
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specifically induces defined double-strand breaks (DSB). Interestingly, the activity of S.
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pyogenes Cas9 is constrained to a protospacer adjacent motif (PAM) of 3 bases (NGG) in the
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genome. However, the Cas9-induced DSB just marks the beginning of the gene editing event.
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The DSB stimulates cellular repair mechanisms, which can lead to a modification in the target
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gene. In most cases, free DNA ends are reconnected by non-homologous end joining (NHEJ). 5
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This can result in a random insertion or deletion of one or more nucleotides that can lead to a
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gene disruption. The second repair mechanism, i.e., homology-directed repair (HDR), can be
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used for the controlled integration of a donor DNA (dDNA). The dDNA needs to share
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homologous flanks with the free DNA ends and can either replace or modify the target gene.16 As
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a result of its simple composition, Cas9-based gene editing has been used in a wide range of
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organisms. In 2015, CRISPR/Cas9 was introduced into filamentous fungi to demonstrate the
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potential of this technique. The delivery of Cas9 as well as the gRNA into the organisms was
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performed with different approaches ranging from plasmids harboring the different genes and
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cassettes to purified Cas9 protein along with the synthesized gRNA.17–26 After the Cas9 mediated
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DSB in the target gene, basic gene disruptions or replacements were conducted. Furthermore, the
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technique was used to remove gene clusters as well as to modify multiple target sites at the same
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time.17,25,26
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In this study, we exploited Cas9-mediated gene editing for the functional reconstitution of tynC in
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the Aspergillus fumigatus strain CEA17 ∆akuB pyrG+. As demonstrated before, this strain was
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proven to carry a nonfunctional tynC allele. Now, we aimed to pinpoint the lack of function of the
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allele by removing the aforementioned single nucleotide insertion. Therefore, we integrated a
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recombinant cas9 expression cassette into the strain CEA17 ∆akuB pyrG+ (akuBKU80 tetON-cas9).
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The expression of cas9 was controlled by a version of the synthetic doxycycline dependent
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(tetON) system27 (Figure 3A). This allowed the regulation of Cas9 activity during editing and
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minimized possible off-target effects previously reported in other organisms.28 Correct
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integration of the cas9 construct in the native, but defective pyrG locus was confirmed by
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Southern blot analysis (Figure S5).
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The bottleneck of gene editing is the selection of positive clones. In recent approaches using
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Cas9-mediated gene editing in filamentous fungi, selection of mutant strains was addressed by
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phenotypic changes of transformants (pigment, growth, resistance) or by integrating a marker
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cassette at the target site. The selection markers were delivered as donor DNA (dDNA) with
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homologous flanks. Thus, the target gene was disrupted by error-prone NHEJ or replaced by the
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dDNA via HDR.17–26 Moreover, the studies are indicating that Cas9-mediated gene replacement
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with marker cassettes is of equal or even higher efficiency compared to established gene deletion
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methods. Nevertheless, the stable integration of a marker in the genome is not an option for
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industrial purposes. Thus, transient selection systems based on self-replicating plasmids have 6
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been applied, which can be lost by culturing without selection pressure.19,22,25,26 Moreover, in
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cases of a modification of a target, markers could hinder the function afterwards. One example
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was shown by Zhang and colleagues (2016) conducting a Cas9-mediated editing attempt in A.
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fumigatus. While integrating a GFP sequence at the N-terminus of the target gene, the selection
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for positive transformants was based on a non-local but random integration of a linear marker
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cassette. At the same time, the authors were modifying the corresponding recognition site for
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Cas9. The change of a single nucleotide in the PAM suppressed further Cas9-mediated DSB at
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the target site, but did not interfere with the gene function. Nevertheless, a counter selection
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based on a phenotypic change of a mutant could have occurred. Hygromycin resistant clones
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without the integrated GFP should be disrupted in the target gene due to NHEJ. Such a scenario,
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i.e., disruption of the gene and occurrence of the corresponding phenotype, was seen in the same
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study as well.25
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In case of the Cas9-mediated editing of tynC shown here, we would not expect any
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distinguishable phenotype on transformation plates or later. Therefore, we preferred the
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introduction of a selectable marker. Nevertheless, the marker had to be spatially separated from
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the tynC locus, to avoid additional alteration of the target locus. Moreover, we wanted to connect
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the activity of the concurrently introduced gRNA with a selection marker. Considering all these
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requirements, we designed a split-marker based selection system, which is functionally connected
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to the expression of the gRNA. The classical split-marker approach was successfully applied to
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fungi to increase the rate of mutants with homologously integrated marker cassettes.29
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Accordingly, our marker of choice, the pyrithiamine resistance cassette (ptrA) of Aspergillus
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oryzae, was divided in two equally sized DNA fragments (Figure 3B and Figure S6A; see also30).
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Both DNA fragments contained 500 bp overlaps to the corresponding part, which would be
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necessary to functionally restore the gene in vivo by the homologous recombination complex.
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The two parts of the split-marker were separated by a 20+3 bp recognition site of the gRNA and
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PAM, which is identical to the target site in the mutated tynC gene in CEA17 ∆akuB pyrG+. The
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split-marker construct was cloned into a plasmid also containing the gRNA cassette (pJW split-
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ptrA tynC; Figure 3B and Figure S6A).
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For the gene editing event the strain akuBKU80 tetON-cas9 was cultivated with doxycycline to
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induce the expression of cas9 (Figure 3A). After the generation of protoplasts of fungal
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mycelium, we used plasmid pJW split-ptrA tynC together with a donor DNA (dDNA) fragment in 7
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a co-transformation experiment. The dDNA was amplified from Af293 encoding the correct
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sequence of tynC at that particular site. As a result of the transformation, pJW split-ptrA tynC
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randomly integrated into the genome. A pre-gRNA was apparently produced. Ribozymes,
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flanking the gRNA sequence were apparently cleaved at the expected size31 (Figure 3B), and the
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matured gRNA interacted with the Cas9 protein (Figure 3C). Cas9 induced DSB in tynC and
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split-ptrA was guided by the tynC specific gRNA (Figure 3C and D). The DSB between the ptrA
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direct repeats induced direct repeat recombination and resulted in a functional ptrA allele (Figure
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3E and F). Hence, cells that contained such functional gRNA-tynC/Cas9 complexes were selected
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on agar plates containing pyrithiamine. At the same time, the tynC locus was likely also cut. The
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derived DNA DSB was repaired by homologous recombination using the provided dDNA as a
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template. This resulted in the desired allele replacement (Figure 3D-F).
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Fungal clones recovered from two individual transformations were successfully transferred to
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pyrithiamine-containing agar plates. Afterwards, 10 clones were tested for the correct editing at
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the target site by sequencing of genomic DNA. Eight of these clones showed the predicted
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reconstitution of the coding sequence and were proven to produce trypacidin (Figure 4 and Figure
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S4). Expression analysis also confirmed the presence of the mRNA of tynC (Figure S1).
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Furthermore, transformation of an akuBKU80 strain lacking Cas9 with the split-marker plasmid did
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not result in growth of colonies (data not shown).
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Taken together, in this study we confirmed that a naturally occurring frameshift in the tynC gene
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blocked the production of trypacidin. First, we could reconstitute the trypacidin biosynthetic
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pathway in a nonproducing strain of A. fumigatus. This was achieved by classical gene deletion
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and complementation to introduce a functional tynC. Then we linked the predicted frameshift
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with the loss of the functional PKS. This was achieved by using CRISPR/Cas9 technology. By
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inducing precisely a site-specific DSB at the targeted locus, one excessive nucleotide in the
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genome was deleted, leading to the production of trypacidin. These results illustrate the great
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potential of CRISPR/Cas9-based site-directed editing of coding sequences in filamentous fungi
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without the need for time-consuming gene deletion, subcloning, in vitro gene modification and
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complementation. Moreover, we presented a more advanced solution for the selection of positive
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transformants after gene editing. The strategy is based on an established selection marker strategy
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but is only functional when the Cas9-gRNA complex is active. This is of importance, especially
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for targets where gene editing events result in transformants without any phenotypic changes. 8
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Further, the gene editing strategy described here, avoids the presence of selectable markers in the
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locus of interest. Apart from studying NP biosynthetic genes, this tool could be useful for
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targeted editing of any functional unit of the genome such as promoters, sites for posttranslational
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modifications in DNA and proteins, or the incorporation of fusion-tags.
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Methods
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Strains and cultivation
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Fungal strains (Table S1) were grown at 37 °C in Aspergillus minimal medium (AMM).32
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Conidia were harvested with water from cultures grown on AMM agar plates at 37 °C for 4 days.
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Conidia concentration was determined by a CASY® TT Cell Counter (OLS Bio, Bremen,
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Germany). 1 x 107 conidia mL-1 were used for pre-culture of transformation as well as stationary
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culture for detection of trypacidin. Escherichia coli DH5α competent cells were used for plasmid
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cloning and propagation. Cultivation was conducted at 37 °C in LB medium supplemented with
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100 µg mL-1 ampicillin (Roth, Karlsruhe, Germany).
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Molecular techniques
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All PCR amplifications were conducted using 2× Phusion High-Fidelity PCR Master Mix (Life
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Technologies, Darmstadt, Germany). For primers, see Table S2. For sequencing of the region of
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interest in tynC, gDNA of fungal strains used in this study was PCR amplified with primers
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oJW0096/97. The resulting PCR fragment was gel-purified (GeneJET Gel Extraction Kit,
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Thermo Fisher Scientific, Darmstadt) and sequenced (LGC Genomics, Berlin, Germany).
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Transformation of A. fumigatus strains was conducted as described before.33 Deletion of tynC
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(AFUB_071800) in CEA17 ∆akuB pyrG+ as well as subsequent complementation with tynCAf293
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(Afu4g14560) was carried out by targeted gene replacement as mentioned before.11 For an
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integration of the cas9 gene in the genome of CEA17 ∆akuB pyrG+ plasmid pJW tetON::cas9::hph
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was targeted to the native but not functional pyrG locus by gene replacement. pJW
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tetON::cas9::hph was obtained by a five fragment (Table S3) Gibson assembly procedure using
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NEBuilder® HiFi DNA Assembly Master Mix according to the manufacturer’s guidelines (New
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England Biolabs, Frankfurt, Germany;34). For this purpose, pUC1835 was linearized by restriction
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digest with SmaI. All other DNA fragments were PCR amplified with primers ensuring at least
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30 bp overlaps to the neighboring fragments of the designed plasmid. DNA fragments #2 and #5
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(each 1 kb), which contain 5’ and 3’ flank of the native pyrG locus, were amplified from gDNA
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of A. fumigatus CEA17 ∆akuB pyrG+ with primers oJW0137/138 and oJW0181/146 respectively.
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Primer pair oJW0139/182 was used to amplify the tetON inducible system (Fragment #3; 2 kb)
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from pSK562 (kindly provided by Prof. S. Krappmann, Erlangen, Germany). A codon-optimized
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cas9 with tef terminator and subsequent hygromycin resistance cassette was amplified at once (6 10
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kb) from pFC33219 with primer pair oJW0183/184, adding an additional SV40 NLS sequence at
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the N-terminus of cas9.
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For transformation, 3 µg of PvuI-linearized pJW tetON::cas9::hph plasmid were added to
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protoplasts of A. fumigatus CEA17 ∆akuB pyrG+. 150 µg mL-1 hygromycin was used as selection
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agent in all following media (Invivogen, Toulouse, France). Colonies of transformants were
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streaked three times on agar plates containing hygromycin. For confirmation of transformants by
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Southern blot, chromosomal DNA of transformants was digested with EcoRV, gel separated and
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transferred to a Nylon membrane (Carl Roth, Karlsruhe, Germany). The membrane was probed
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with a DIG-labeled DNA fragment, which was amplified from gDNA of parental strain using
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primers oJW0137/138.
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The split-marker strategy for gene editing of tynC locus is based on pJW split-ptrA tynC and an
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expressed cas9 in the transformant strains. Gibson assembly of pJW split-ptrA tynC was achieved
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with four PCR-amplified DNA fragments (see Table S3) and the procedure as described for pJW
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tetON::cas9::hph, ensuring 30 bp overlaps to the neighboring DNA fragments. Fragment #1 (ampR
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+ ptrA part 1; 3.2 kb) and fragment #2 (ptrA part 2 + ori; 1.9 kb) were amplified from pSK27536
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with primers oJW0228/197 and oJW0198/199, respectively. gRNA cassette with tynC-specific
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spacer was amplified in two DNA fragments (#3 and #4) from pFC33419 with primers
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oJW0200/185 and oJW0186/211. The gRNA spacer was selected manually. The sequence targets
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the site of the single nucleotide insertion in tynC of CEA17 ∆akuB pyrG+. The induced DSB
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would be next to the target site, but cannot target the tynC of Af293. BLASTN alignment did not
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find any other targets which could affect the trypacidin biosynthesis.
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The generated split-ptrA gRNA plasmid also enables targeting of any gene of interest. Using
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overlapping primers, the spacer of the gRNA construct as well as the compatible protospacer site
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in the split-marker construct can be replaced. The designed separation of ori and ampR to both
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DNA fragments ensures the expected recombination during plasmid assembly and avoids false-
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positive clones upon transformation of E. coli (Figure S6).
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Strain akuBKU80 tetON-cas9 was used for gene editing of the tynC locus. The expression of the
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cas9 gene was induced by 5 µg mL-1 doxycycline (Sigma-Aldrich, Taufkirchen, Germany) added
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to the fungal pre-culture before transformation. For the design of the dDNA, we chose the 880 bp
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DNA fragment which was also amplified for sequencing with primers oJW0096/97 (see above). 11
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The fragment covers the predicted single nucleotide insertion in CEA17 ∆akuB pyrG+, but no
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further variation in any of the strains studied. The dDNA was amplified from strain Af293
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gDNA. A further aspect of the dDNA is the size of the homology arms. Previous studies showed
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that dDNA for HDR after Cas9-mediated DSB can range from 39 to 1000 bp. The length of the
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homology arms for our purpose ranged between 400-500 bp.
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According to the transformation protocol, protoplasts of akuBKU80 tetON-cas9 were transformed
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with 1 µg pJW split-ptrA tynC as well as 1 µg dDNA. Selection of transformants and three
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streak-outs of resulting transformants were carried out on agar plates containing 0.1 µg mL-1
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pyrithiamine (Sigma-Aldrich, Taufkirchen, Germany). For the proof of site-directed editing,
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gDNA of transformants was PCR amplified with primers oJW0096/97 and sequenced as
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mentioned before.
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Extraction and analysis of trypacidin
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Fungal stationary cultures grown at 37 °C for 3 days were homogenized (Ultra-Turrax, IKA,
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Staufen, Germany) and subsequently extracted with 2 volumes (v/v) ethyl acetate. After
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dehydration of the organic phase with anhydrous sodium sulfate, samples were separated by filter
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paper and concentrated by rotary evaporator. Crude extracts were dissolved in 1 mL of methanol
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and filtered again with a 0.2 µm PTFE filter (Carl Roth, Karlsruhe, Germany). LC-MS analysis
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was conducted as mentioned before.11 Results were compared to an authentic trypacidin standard
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(Biomol, Hamburg, Germany).
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Expression analysis
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Fungal stationary cultures grown at 37 °C for 24 h. Mycelium was collected and frozen in liquid
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nitrogen. Samples were ground to powder using mortar and pestle. Total RNA was extracted with
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RNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
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10 µg of total RNA was treated with DNase I using the TURBO DNA-free™ Kit (Ambion,
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Darmstadt, Germany). 5 µg DNaseI-treated RNA was used for cDNA synthesis, which was
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conducted with RevertAid Reverse Transcriptase (Thermo Scientific, Schwerte, Germany).
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Expression of tynC and the housekeeping gene act1 was detected in a PCR reaction with 35
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cycles using gene specific primers (see Table S2). For the detection of tynC we used 2 µL for
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act1 1 µL of the cDNA template and primer pairs oJW0434/435 and oJW0436/437, respectively.
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Figure Legends
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Figure 1. Single nucleotide insertion in the PKS-encoding gene tynC of A. fumigatus strain
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CEA10 is predicted to be responsible for the lack of trypacidin production. (A) Sequencing
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of tynC in strains Af293, CEA10 and its descendant akuBKU80 confirmed single adenosine
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insertion at position 3881 in the latter two strains (arrows). Single nucleotide insertion leads to a
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frameshift with D1248E exchange and subsequent stop codon in the predicted DNA sequence
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(red box). Premature stop codon eliminates PT (product template) and ACP (acyl carrier protein)
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domains from TynC and leads to a truncated protein with SAT (starter-unit ACP transacylase),
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KS (β-ketoacyl synthase) and AT (ACP transacylase) domain. (B) Extracted ion chromatograms
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(EIC: m/z 345 [M + H]+) of 3 days old stationary cultures.
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Figure 2. Trypacidin production in akuBKU80 was reconstituted by complementation with a
315
functional tynC gene. EIC (m/z 345 [M + H]+) of 3 days old stationary cultures of akuBKU80,
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akuBKU80 ∆tynC, akuBKU80 tynCAf293 comp(lemented) and trypacidin standard (see also Figure S4
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for MS2).
318
Figure 3. Overview of strategy for targeted gene editing with split-marker approach. (A) A.
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fumigatus strain harboring the cas9 gene under the control of an inducible tetON promoter was
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created (akuBKU80 tetON-cas9). (B) Then, a plasmid comprising split-marker construct (dark-blue)
321
and gRNA construct (details see Figure S6) was used to transform strain akuBKU80 tetON-cas9. By
322
adding doxycycline prior to transformation (A), the fungal recipient strain expressed Cas9
323
nuclease gene. gRNA construct was expressed after integration into the genome (B). Pre-gRNA
324
matured by self-cleaving ribozymes (yellow) on both sites of the pre-gRNA31, yielding the
325
mature gRNA (~100 bp) with recognition unit of target site (spacer; pink) and scaffold (black).
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(C) gRNA and Cas9 assembled and bound to the recognition site (protospacer, pink) in the
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genome at the tynC locus as well as in the integrated split-marker. (D) Due to the adjacent PAM
328
site Cas9 induced double strand breaks (DSB). (E) Homologous recombination of DSB occurred
329
at the target gene locus, due to the use of the provided donor DNA (dDNA), and in fragmented
330
split-marker, exploiting the 500 bp nucleotide repetitions (white stripes). (F) Selection of edited
331
PKS gene-containing strains based on the reconstitution of functional selection marker gene.
332
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Figure 4. Trypacidin production in akuBKU80 was reconstituted by CRISPR/Cas9-mediated
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elimination of one excessive adenosine in the endogenous gene. (A) EIC (m/z 345 [M + H]+)
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shows presence of trypacidin in 3-days old stationary cultures of Af293 and akuBKU80 tetON-cas9
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tynC+, but not of akuBKU80 tetON-cas9 (see also Figure S4 for MS2). (B) Sequencing of tynC in
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the shown strains confirmed elimination of single adenosine (indicated by arrows) in akuBKU80
338
tetON-cas9 tynC+.
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Figure 2
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Figure 3
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References
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(1) Scharf, D. H., Heinekamp, T., and Brakhage, A. A. (2014) Human and plant fungal pathogens: the role of secondary metabolites. PLoS Pathog. 10, e1003859.
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(2) Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., Kim, H. S., Arroyo, J., Berriman, M., Abe, K., Archer, D. B., Bermejo, C., and others. (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151–1156. (3) Fedorova, N. D., Khaldi, N., Joardar, V. S., Maiti, R., Amedeo, P., Anderson, M. J., Crabtree, J., Silva, J. C., Badger, J. H., Albarraq, A., and others. (2008) Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. PLoS Genet. 4, e1000046. (4) Brakhage, A. A. (2013) Regulation of fungal secondary metabolism. Nat. Rev. Microbiol. 11, 21–32. (5) Netzker, T., Fischer, J., Weber, J., Mattern, D. J., Kӧnig, C. C., Valiante, V., Schroeckh, V., and Brakhage, A. A. (2015) Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters. Front. Microbiol. 6, 299. (6) Mattern, D. J., Valiante, V., Unkles, S. E., and Brakhage, A. A. (2015) Synthetic biology of fungal natural products. Front. Microbiol. 6, 775. (7) Kato, N., Suzuki, H., Okumura, H., Takahashi, S., and Osada, H. (2013) A point mutation in ftmD blocks the fumitremorgin biosynthetic pathway in Aspergillus fumigatus strain Af293. Biosci., Biotechnol., Biochem. 77, 1061–1067. (8) Frisvad, J. C., Rank, C., Nielsen, K. F., and Larsen, T. O. (2009) Metabolomics of Aspergillus fumigatus. Med. Mycol. 47, S53–S71. (9) Nemec, P. B. J. E. L. (1963) Antiprotozoal antibiotics. I -Method of specific screening. J. Antibiot., Ser. A 16, 155–156. (10) Gauthier, T., Wang, X., Dos Santos, J. S., Fysikopoulos, A., Tadrist, S., Canlet, C., Artigot, M. P., Loiseau, N., Oswald, I. P., and Puel, O. (2012) Trypacidin, a spore-borne toxin from Aspergillus fumigatus, is cytotoxic to lung cells. PloS One 7, e29906. (11) Mattern, D. J., Schoeler, H., Weber, J., Novohradská, S., Kraibooj, K., Dahse, H.-M., Hillmann, F., Valiante, V., Figge, M. T., and Brakhage, A. A. (2015) Identification of the antiphagocytic trypacidin gene cluster in the human-pathogenic fungus Aspergillus fumigatus. Appl. Microbiol. Biotechnol. 99, 10151-10161. (12) Throckmorton, K., Lim, F. Y., Kontoyiannis, D. P., Zheng, W., and Keller, N. P. (2015) Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus. Environ. Microbiol. 18, 246-259. (13) Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics 24, 18
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637–644. (14) Krappmann, S. (2014) Genetic surgery in fungi: employing site-specific recombinases for genome manipulation. Appl. Microbiol. Biotechnol. 98, 1971-1982. (15) Kim, H., and Kim, J.-S. (2014) A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321-334. (16) Maeder, M. L., and Gersbach, C. A. (2016) Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430-446. (17) Liu, R., Chen, L., Jiang, Y., Zhou, Z., and Zou, G. (2015) Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discovery 1, 15007. (18) Arazoe, T., Miyoshi, K., Yamato, T., Ogawa, T., Ohsato, S., Arie, T., and Kuwata, S. (2015) Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotechnol. Bioeng. 112, 2543–2549. (19) Nødvig, C. S., Nielsen, J. B., Kogle, M. E., and Mortensen, U. H. (2015) A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PloS One 10, e0133085. (20) Matsu-ura, T., Baek, M., Kwon, J., and Hong, C. (2015) Efficient gene editing in Neurospora crassa with CRISPR technology. Fungal Biol Biotechnol. 2:4, 1–7. (21) Fuller, K. K., Chen, S., Loros, J. J., and Dunlap, J. C. (2015) Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryotic Cell 14, 1073–1080. (22) Schuster, M., Schweizer, G., Reissmann, S., and Kahmann, R. (2015) Genome editing in Ustilago maydis using the CRISPR-Cas system. Fungal Genet. Biol. 89, 3-9. (23) Fang, Y., and Tyler, B. M. (2016) Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol. Plant Pathol. 17, 127–139. (24) Katayama, T., Tanaka, Y., Okabe, T., Nakamura, H., Fujii, W., Kitamoto, K., and Maruyama, J. (2015) Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol. Lett. 38, 637-642. (25) Zhang, C., Meng, X., Wei, X., and Lu, L. (2016) Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet. Biol 86, 47–57. (26) Pohl, C., Kiel, J. A., Driessen, A. J., Bovenberg, R. A., and Nygård, Y. (2016) CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth. Biol. 5, 754-764. (27) Helmschrott, C., Sasse, A., Samantaray, S., Krappmann, S., and Wagener, J. (2013) Upgrading fungal gene expression on demand: Improved systems for doxycycline-dependent silencing in Aspergillus fumigatus. Appl. Environ. Microbiol. 79, 1751–1754. 19
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(28) O’Geen, H., Abigail, S. Y., and Segal, D. J. (2015) How specific is CRISPR/Cas9 really? Curr. Opin. Chem. Biol. 29, 72–78. (29) Kück, U., and Hoff, B. (2010) New tools for the genetic manipulation of filamentous fungi. Appl. Microbiol. Biotechnol 86, 51–62. (30) Schafferer, L., Beckmann, N., Binder, U., Brosch, G., and Haas, H. (2015) AmcA—a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis. Front. Microbiol 6, 252. (31) Gao, Y., and Zhao, Y. (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349. (32) Brakhage, A. A., and Van den Brulle, J. (1995) Use of reporter genes to identify recessive trans-acting mutations specifically involved in the regulation of Aspergillus nidulans penicillin biosynthesis genes. J. Bacteriol. 177, 2781–2788. (33) Weidner, G., d’ Enfert, C., Koch, A., Mol, P. C., and Brakhage, A. A. (1998) Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′-monophosphate decarboxylase. Curr. Genet. 33, 378–385. (34) Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345. (35) Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103–119. (36) Szewczyk, E., and Krappmann, S. (2010) Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryotic Cell 9, 774–783.
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Supporting information
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Figure S1, expression analysis of tynC; Figure S2, alignment of DNA sequences; Figure S3,
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Southern blot of ∆tynC and tynCAf293 complemented; Figure S4, MS2 fragmentation pattern for
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m/z 345 [M + H]+; Figure S5, Southern blot of akuBKU80 tetON-cas9; Figure S6, plasmid map of
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split-ptrA tynC; Table S1, list of strains; Table S2, list of primers; Table S3, list of plasmids.
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Acknowledgements
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Carmen Schult is gratefully acknowledged for excellent technical assistance. This research was
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supported by the BMBF-funded project DrugBioTune in the frame of InfectControl2020, the
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excellence graduate school Jena School for Microbial Communication and the DAAD RISE
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Program.
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Competing interests
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The authors declare no competing interests.
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graphical abstract graphical abstract 169x76mm (200 x 200 DPI)
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Single nucleotide insertion in the PKS-encoding gene tynC of A. fumigatus strain CEA10 is predicted to be responsible for the lack of trypacidin production. (A) Sequencing of tynC in strains Af293, CEA10 and its descendant akuBKU80 confirmed single adenosine insertion at position 3881 in the latter two strains (arrows). Single nucleotide insertion leads to a frameshift with D1248E exchange and subsequent stop codon in the predicted DNA sequence (red box). Premature stop codon eliminates PT (product template) and ACP (acyl carrier protein) domains from TynC and leads to a truncated protein with SAT (starter-unit ACP transacylase), KS (β-ketoacyl synthase) and AT (ACP transacylase) domain. (B) Extracted ion chromatograms (EIC: m/z 345 [M + H]+) of 3 days old stationary cultures. Figure 1 177x82mm (150 x 150 DPI)
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Trypacidin production in akuBKU80 was reconstituted by complementation with a functional tynC gene. EIC (m/z 345 [M + H]+) of 3 days old stationary cultures of akuBKU80, akuBKU80 ∆tynC, akuBKU80 tynCAf293 comp(lemented) and trypacidin standard (see also Figure S4 for MS2). Figure 2 83x77mm (150 x 150 DPI)
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Overview of strategy for targeted gene editing with split-marker approach. (A) A. fumigatus strain harboring the cas9 gene under the control of an inducible tetON promoter was created (akuBKU80 tetON-cas9). (B) Then, a plasmid comprising split-marker construct (dark-blue) and gRNA construct (details see Figure S6) was used to transform strain akuBKU80 tetON-cas9. By adding doxycycline prior to transformation (A), the fungal recipient strain expressed Cas9 nuclease gene. gRNA construct was expressed after integration into the genome (B). Pre-gRNA matured by self-cleaving ribozymes (yellow) on both sites of the pre-gRNA31, yielding the mature gRNA (~100 bp) with recognition unit of target site (spacer; pink) and scaffold (black). (C) gRNA and Cas9 assembled and bound to the recognition site (protospacer, pink) in the genome at the tynC locus as well as in the integrated split-marker. (D) Due to the adjacent PAM site Cas9 induced double strand breaks (DSB). (E) Homologous recombination of DSB occurred at the target gene locus, due to the use of the provided donor DNA (dDNA), and in fragmented split-marker, exploiting the 500 bp nucleotide repetitions (white stripes). (F) Selection of edited PKS gene-containing strains based on the reconstitution of functional selection marker gene.
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Figure 3 131x281mm (100 x 100 DPI)
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Trypacidin production in akuBKU80 was reconstituted by CRISPR/Cas9-mediated elimination of one excessive adenosine in the endogenous gene. (A) EIC (m/z 345 [M + H]+) shows presence of trypacidin in 3-days old stationary cultures of Af293 and akuBKU80 tetON-cas9 tynC+, but not of akuBKU80 tetON-cas9 (see also Figure S4 for MS2). (B) Sequencing of tynC in the shown strains confirmed elimination of single adenosine (indicated by arrows) in akuBKU80 tetON-cas9. Figure 4 174x74mm (150 x 150 DPI)
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