Fig 2. Construction of new CRISPR-Cas9 vectors for directed mutagenesis of filamentous fungi.

Construction of fungal CRISPR-Cas9 vectors with variable sgRNA genes controlled by gpdA promoter and trpC terminator (no DNA elements are drawn to scale). The vector backbone for construction of new Fungal vectors for Cas9 induced genetic engineering are derived from the plasmid series pFC330-333. Sticky ends for USER cloning are achieved by opening the PacI/Nt.BbvCI USER cassette of pFC330-333 by the concerted action of restriction enzymes PacI and Nt.BbvCI (left side of panel). The two PCR fragments necessary for construction of the sgRNA gene, are both obtained by using pFC334 as template (right side of panel). This vector contains a protospacer for targeting yA (in light green), which is represented by 20 Ns indicating that it is not intended to match the primer; and in principle could be any sequence. The sections of the sgRNA gene encoding the variable parts of the transcript, the 20 bases of the protospacer (in light green) and the reverse complementing 6 bases of HH (in light blue), are introduced via tails added to the ends of the two primers that define the down- and upstream ends of the two PCR fragments, respectively. The position of the resulting inverted repeat located in the variable regions is indicated by blue arrows (top of panel). After amplification, the two PCR fragments are fused and inserted into vector pFC330-333 (Four variants exist) by USER cloning in a single step. For this purpose, each PCR fragment is generated by primers containing a tail with a uracil base (in purple). Elimination of the uracil bases in the PCR fragments by Uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII (USER Enzyme) results in the production of pairwise complementary overhangs at the ends of all fragments allowing selected ends to be fused in a directional manner. For simplicity, all complementary ends are visualized in the same color.