Vector construction

PCR fragments were amplified using the PfuX7 polymerase [21] with primers purchased from Integrated DNA Technology, Belgium (S1 Table). Construction of vectors was carried out by Uracil-Specific Excision Reagent (USER) fusion of PCR fragment into compatible plasmids [22]. The deletion plasmids pD-hyg-talA and pD-hyg-albA were constructed by amplification of approximately 2-kb up- and downstream fragments followed by cloning into two distinct PacI/Nt.BbvCI USER cassettes located on each side of the hygromycin resistance gene. The sgRNA was introduced into the CRISPR-Cas9 vector pFC330 via the tails of two primers as described by Nødvig et al. [4]. Plasmids were purified using the GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich), and verified by restriction analysis. A list of all plasmids from this study is presented in S2 Table. Deletions were achieved using the CRISPR-Cas9 system described by Nødvig et al. [4]. A circular deletion plasmid (gene-targeting substrate) was co-transformed with an AMA1-based CRISPR-Cas9 vector containing the guide RNA and the Streptococcus pyogenes cas9 gene codon optimized for A. niger. The CRISPR-Cas9 vector also contained the pyrG auxotrophic marker; however, only the deletion plasmid, containing the hygromycin resistance gene, was selected for during transformation.