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Official Journal of the Japan Wood Research Society

Fig. 3 | Journal of Wood Science

Fig. 3

From: Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection

Fig. 3

Confirmation of transient Cas9 expression and genome editing. a PCR amplification of cas9 gene fragment using primer sets Cas9check_F2 and Cas9check_R2. Lane M, 1-kb ladder (Nippon gene); lane P, pCcPef3-126-fcy1sg2 (positive control); lane W, parental strain PC9 (negative control); lanes1, Pef3-5. b A schematic diagram of the fcy1 loci in the PC9 parental strain with gRNA recognition sites. The dash lines indicate the region amplified by genomic PCR. Black arrows display the primer pairs used for the PCR experiments and Sanger sequencing. c PCR amplification of a target site of gRNA in the fcy1 using primer set TB9 and TB10. Lane M, 1-kb ladder (Nippon gene); lane W, parental strain PC9; lanes1, Pef3-5. d DNA sequencing to identify mutations of fcy1 in Pef3-5 strain. For highlights in the nucleotide sequence: yellow shades indicate gRNA, green shades indicate a protospacer adjacent motif (PAM) sequence, and dash lines indicate deletion by insertion of a plasmid sequence. e Insertion sequence in fcy1 locus of Pef3-5 strain revealed by Sanger sequencing. For highlights in the nucleotide sequence: blue shades indicate a sequence derived from hph, magenta shades indicate a sequence derived from CcTub_ter, and under lines indicate sequence derived from pCcPef3-126-fcy1sg2

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