Fig. 2: Identification and evaluation of neutral loci for gene expression.

A A computational approach was used to identify intergenic regions with high expression of surrounding genes. The highest expressing regions were selected as promising neutral loci for further experimental evaluation. B Targeting plasmids were designed with the 5′ and 3′ homology arms as well as the specific 300 bp sequence for the locus of interest. The plasmid harbors a GFP-expression cassette driven by the constitutive pTEF1 promoter and terminated by the commonly used TamyB terminator, both from A. oryzae. The plasmids were cloned in E. coli and were linearized using PCR to create linear fixing templates that target the locus of interest. C Flow cytometry of conidia constitutively expressing pTEF1 was used to evaluate expression strength at the neutral locus of interest and determine their suitability for engineering efforts. A representative microscopy image showing GFP expression from A. oryzae conidia is shown. Scale bar = 25 µm. The flow cytometry figure was created was created using BioRender (http://BioRender.com). D Integration efficiency of GFP-expression cassette at neutral loci. All loci except for chro3_1 displayed a high efficiency of integration (>50%), as assessed by PCR. We could not detect insertion at chro3_1 by PCR. E GFP expression (expressed as Mean Equivalents of Fluorescein, or MEFL) across neutral loci. All loci except for chro4_2 displayed expression levels above the background strain (RIB40) and were higher than the amyA locus, which was included as a positive control to validate the method. Results are average and standard error of the mean (SEM) of three biological replicates.