Fig. 2

Marker-free genome editing by transient expression of Cas9, gRNA, and hph. a Schematic diagram of marker-free genome editing. b The plasmid map of pCcPef3-126-fcy1sg2. pUC ori: origin for Escherichia coli, KmR: kanamycin resistance gene, AbHSP26_ter: terminator from hsp26 gene in Agaricus bisporus. NLS: Nuclear localization signal, fcoCas9: Fungal and plant codon optimized Cas9 [10], CcEF3_pro: Promoter from ef3 gene in Coprinopsis cinerea, CcU6_pro: U6 promoter from C. cinerea, CctubB_pro or ter: Promoter or terminator from tubulin gene in C. cinerea, hph: hygromycin phosphotransferase. c PCR amplification of hph fragment using primer set hph_F and hph_R. Lane M, 1-kb ladder (Nippon gene); lane P, pPHT1 (positive control); lane W, parental strain PC9 (negative control); lanes 1–6, Pef3-1–6 strains. Hyg^R: hygromycin B resistant, Hyg^S: hygromycin B sensitive