Fig. 3

Overexpression of the xyr1-A824V gene at the ace1 locus in the T. reesei genome through homologous recombination mediated by the tRNA−gRNA array-based CRISPR−Cas9 editing system. A Schematic diagram of the xyr1-A824V overexpression process. After transformed to the parental strain QM53, two sgRNAs (sgRNA-ace1U and sgRNA-ace1D) were co-expressed from the tRNA−gRNA array cassette named as 5StsgRNA (ace1*2). Subsequently, Cas9 endonucleases were recruited to the target sites of ace1 by sgRNA-ace1U and sgRNA-ace1D to generate two double-strand breaks (DSBs, ▲). The broken chromosome can be repaired by homologous recombination (HR) pathway using donor DNA (the xyr1-A824V expression cassette). The cassette named as dgXYR1-ace1 contains the gpdA promoter (PgpdA), the gene coding region of xyr1-A824V, the trpC terminator TtrpC, the upstream homologous arm of ace1 (U-ace1) and the downstream homologous arm of ace1 (D-ace1). In addition, the cassette named as dcXYR1-ace1 has only the cdna1 promoter Pcdna1 that differs from dgXYR1-ace1. The final xyr1-A824V overexpressing strains constructed with these two cassettes were named as QA1Xg and QA1Xc respectively. B, C Transcript levels of xyr1 in the xyr1-A824V overexpressing strains QA1Xg, QA1Xc and the parental strain QM53 at 72 h under Avicel culture conditions and glucose culture conditions, respectively. Data are the Mean ± SD of the results from three independent experiments. Significant differences were analyzed using t test. (*p < 0.05, **p < 0.01.)