## 3 Results
### Endoglucanase Integration and Activity  
To ascertain if different intergenic loci would lead to differences in heterologous protein production levels, we targeted single locus integration of _T.r.eg2_ to different chromosomal sites in diploid and haploid _S. cerevisiae_ strains. The intergenic chromosomal target sites selected were previously shown to support heterologous protein production at high levels [33]. After the _T.r.eg2_ gene was confirmed to be present in the transformed MH1000 and M1744 strains via activity screening and PCR, strains were cultured to quantify their endoglucanase activities, as shown in Figure 2. The parental strain was included as negative  
Figure 1: A schematic overview of the CRISPR-Cas9 systems used to transform a diploid industrial and a haploid laboratory _S. cerevisiae_ strain. (**a**) Schematic illustration of the two-plasmid CRISPR system. A low-copy replicative (CEN/ARS-containing) plasmid contained the _cas9_ gene and the selection marker for CloNAT resistance, and a multi-copy plasmid (2 ) contained the gRNA expression cassette and the selection marker for G418 resistance. (**b**) Schematic illustration of the one-plasmid CRISPR system. A multi-copy (2 ) plasmid contained the _cas9_ gene, a gRNA cassette, and a selection marker for G418 resistance. (**c**) The CRISPR system targeted chromosomal intergenic sites for the integration of different genomic repair expression cassettes for gene editing. “Delta” represents the repeated Ty delta elements dispersed in the yeast genome that allows multi-target integration. Delta sequences are referred to by the symbol  elsewhere. (**d**) The repair DNA cassettes contained reporter genes flanked by the _ENO1_ promoter, _ENO1_ terminator, and 40-bp homology arms, targeting integration to the various genomic sites. control and resulted in no activity, as expected. It was shown that both haploid and diploid _S. cerevisiae_ strains had significantly lower activities when the gene was targeted to Ch10 or Ch11, compared to Ch12 (\(p\leq 0.05\)). At 72 h of cultivation, the highest activity for haploid M1744 strains was 160 U/gDCW for the Ch12 targeted strain, and the lowest activity was 40 U/gDCW for the Ch10 targeted strain. The highest activity observed in diploid MH1000 strains was 48 U/gDCW for the Ch12 targeted strain, and the lowest activity observed was 18 U/gDCW for the Ch11 targeted strain. Based on these findings, the haploid M1744 strain also had higher EG activities in all the targeted chromosome sites than the diploid MH1000 strain on a per DCW basis. Due to the differences in activity observed, we were interested in determining the number of _T.reg2_ gene copies integrated into the genome of each transformant to disambiguate locus and copy number effects on expression level. This was determined using qPCR (Table 3). We expected the _S. cerevisiae_ M1744 transformants to have one copy of the _T.reg2_ gene because they are haploid with only one set of chromosomes and, thus, one target site. Since MH1000 _S. cerevisiae_ strains are diploid and have two sets of chromosomes, we expected them to have at least two copies of the _T.reg2_ gene. For targeting chromosomes X and XI, qPCR results matched our expectations as we confirmed one copy of _T.reg2_ in the M1744 yeast strains and two copies in the MH1000 yeast strains. However, we were unable to accurately determine the copy number of _T.reg2_ genes targeted to Ch12 from qPCR in MH1000-Ch12-EG2. Furthermore, the M1744-Ch12-EG2 strain was unexpectedly found to contain three copies of the gene. Evaluating the position of integration in these strains using standard PCR also indicated possible off-target integration. Our CRISPR systems achieved integration efficiencies of 55-65% for transformants expressing _T.reg2_ at different sites in the yeast genome. Figure 2: Endoglucanase activity profiles of recombinant yeast strains after 48 and 72 h cultivation. (**a**) The activity of EG2 producing haploid strains on CMC. (**b**) The activity of EG2 producing diploid strains on CMC.