Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01

CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes. Based on the above method, providing ideal neutral integration sites can ensure the reliable, stable, and high expression of target genes. In this study, we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS. The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension, and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs (Sites) for CRISPR/Cas9-mediated homology-directed recombination. PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%. Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F. venenatum TB01. These results indicate the above designed neutral sites have the potential to accelerate the development of F. venenatum TB01 through overexpression of target genes in metabolic engineering.