Fig. 3
Editing of Poria cocos squalene epoxidase gene by a dual-sgRNA-directed CRISPR/Cas9 system through PEG-mediated protoplast transformation. WT denotes the wild-type strain, Tr represents transformed un-knocked strains, Mu1-Mu5 are mutants. A Verification of the sgRNA1 deletion strain by PCR. M, DL 2 000 DNA marker; 898 bp represents transfer-in plasmid using primers for validation and 616 bp for validation using knockout primers. B Screening and re-selection of ergosterol-resistant mutants after sgRNA1 targeting SE were inserted into transformant pFC332-U6-2-sgRNAPcSE-Cas9. C DNA sequences of the target fragment of the sgRNA1 knockout strains. The red and green boxes indicate the PAM and guide sequence, respectively; + indicates inserted fragments,—indicates deleted fragments; Mu1, 1 bp deletion; Mu2, 1 bp insertion and 2 bp deletion; Mu3, 2 bp deletion; Mu4, 1 bp substitution and 2 bp deletion; Mu5, 4 bp deletion, 1 bp substitution and 3 bp insertion, respectively