CRISPR/Cas9 plasmids construction

All plasmids and primers used in this study are listed in the supplementary data (Additional file 1: Table S1, Additional file 6: Table S2). In silico work was performed on FASTA files obtained from JGI [65]. Plasmid construction was based on earlier work performed in A. niger [50]. A detailed protocol on the CRISPR/Cas9 plasmid construction and subsequent transformations in P. variotii and P. roqueforti can be found in the supplementary data (Additional file 7: Protocol S1). Briefly, the plasmids pTLL108.1 and pTLL109.2 were used as templates for creating the 5′ flank and 3′ flank of the sgRNA respectively. After fusion PCR using the pTE1_for and pTE1_rv primers, a PacI (Fermentas, Thermo Scientific™) digestion on the purified PCR product was performed O/N. The PacI digested sgRNA was ligated into a PacI digested and dephosphorylated pFC332 plasmid and subsequently cloned into E. coli DH5α. The ampicillin resistant colonies were grown under selective pressure overnight and miniprepped (GeneJET Plasmid Miniprep Kit, Thermo Scientific™), after which restriction analysis was done with SacII (Fermentas, Thermo Scientific™) to check for correct insertion of the sgRNA. Lastly, sequencing was performed as a final check to ensure correct sgRNA is present in the newly constructed plasmid.