A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in .

is a highly versatile fungal strain utilized in industrial production. The expression levels of recombinant genes in can be enhanced by increasing the copy number. Nevertheless, given the prolonged gene editing cycle of , a "one-step" strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage. In our previous study, a visual multigene editing system (VMS) was designed to knock out five genes, employing a tRNA-sgRNA array that includes the pigment gene and the target genes. Building upon this system, hybrid donor DNAs (dDNAs) were introduced to establish a clustered regularly interspaced short palindromic repeats (CRISPR)-based multiplex integration toolkit. Firstly, a CRISPR-Cas9 homology-directed repair (CRISPR-HDR) system was constructed in by co-transforming the CRISPR-Cas9 plasmid (with a highly efficient sgRNA) and the dDNA, resulting in precise integration of recombinant xylanase gene into the target loci (the β-glucosidase gene , the amylase gene , and the acid amylase gene ). Subsequently, the length of homology arms in the dDNA was optimized to achieve 100% editing efficiency at each of the three gene loci. To achieve efficient multiplex integration in , the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci (, , , and ). Hybrid dDNAs were then employed for repair, including dDNA1-3 (containing expression cassettes without selection markers) and dDNA (for knockout). Among the obtained white colonies (RLM2'), 23.5% exhibited concurrent replacement of the , , and genes with (three copies). Notably, the xynA activity obtained by simultaneous insertion into three loci was 48.6% higher compared to that obtained by insertion into only the locus. Furthermore, this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and lipase CALB. Hence, the combined application of VMS and the CRISPR-HDR system enabled the simultaneous application of multiple selection markers, facilitating the rapid generation in the cell factories.