Fig. 5.

Schematic representation of CRISPR/Cas9-mediated gene targeting in V. dahliae. a Both SpCas9 and sgRNA cassettes were introduced into the same, AMA1-containing plasmid. The sgRNA was flanked by the hammerhead (HH) and hepatitis delta virus (HDV) ribozymes, which enable the release of the mature sgRNA after their self-catalysis (scissors indicate cleavage points). b Homologous repair substrates consist of either a fluorescent protein gene (for gene tagging) or an antibiotic-resistance cassette (for gene disruption), flanked by homologous arms of variable length. c Co-transformation of the AMA1-containing plasmid with the homologous repair substrate for gene tagging or disruption. The Cas9/sgRNA complex induces in the genomic locus targeted DSBs (scissors), which can be repaired using the homologous repair substrate to result in seamless gene tagging or gene disruption. In the latter case, stop codons (asterisk) were introduced in the homologous arm downstream of the antibiotic-resistance cassette, to ensure the knockout of the gene (example shown: V. dahliae atg8)