Fig. 4

Construction of marker free repair DNA fragment. Amplification of regions upstream (5′ flank) and downstream (3′ flank) of a gene of interest (GOI): Primer 1 and Primer 3 are used to amplify the 5′ flank, typically directly upstream from the start codon of the GOI. Primer 2 and Primer 4 are used to amplify the 3′ flank, just downstream of the ORF stop codon. Addition of 20 bp reverse complement sequence of Primer 3 to the 5′ end of Primer 2 ensures overlap between the 5′ and 3′ flanks necessary for fusion of the two flanks to construct the marker free repair DNA fragment. Upon introduction of the marker free repair DNA fragment to the fungal cell, repair of the double strand break (DSB) induced by a Cas9–sgRNA complex is possible by homology directed repair (HDR) at the site of the GOI