Fig. 7

Simultaneous expression of the gox gene at the cbh1 locus and deletion of the cbh2 gene in the T. reesei genome by the tRNA−gRNA array-based CRISPR−Cas9 system. A Schematic diagram of the simultaneous multi-gene editing process. After transformed to the parental strain QM53, four sgRNAs (sgRNA-cbh1U, sgRNA-cbh1D, sgRNA-cbh2U and sgRNA-cbh2D) were co-expressed from the tRNA-sgRNA array cassette named as 5StsgRNA (cbh1*2-cbh2*2). Subsequently, Cas9 endonucleases were recruited to target sites of the cbh1 and cbh2 gene by sgRNA-cbh1U, sgRNA-cbh1D, sgRNA-cbh2U and sgRNA-cbh2D, generating double-strand breaks (DSB, ▲). The broken cbh1 gene could be repaired by homologous recombination (HR) pathway using the donor DNA (the gox expression cassette). The dDNA named as dGOD-cbh1 contains the cbh1 promoter Pcbh1, the CBH1 signal peptide (sp), the gene coding region of gox and the cbh1 terminator Tcbh1. In addition, the broken cbh2 gene could be repaired by non-homologous end-joining (NHEJ) to complete the deletion process. The final strain was named as QGOD. B Qualitative evaluation for the ability to secrete glucose oxidase using the plates where lactose was served as the sole carbon source and dye liquor was dumped containing o-dianisidine, HRP and glucose. C The transcript level of the gox gene in the strains QGOD and QM53. D The transcript levels of cbh1 and cbh2 in the strains QGOD and QM53. All values were normalized to actin expression under same conditions. Data are the Mean ± SD of the results from three independent experiments. ND = No Detection