## 3 Results
### Endoglucanase Integration and Activity
Values obtained were normalized with the dry cell weight of each specific yeast strain. The M1744 and MH1000 parental strains were used as negative control reference strains. The values given are the mean values of enzyme assays conducted in triplicate. Error bars indicate the standard deviation from the mean value for each strain. Subsequently, we targeted _T.r.eg2_ to the delta-sequences for multi-copy integration of the gene in both haploid and diploid _S. cerevisiae_ strains to potentially increase the integrated gene copy number. After confirmation of transformation, EG activities of the delta-integrated transformants were determined (Figure 3). As expression levels can vary significantly between different chromosomal regions [34], we tested eight positive transformants for each strain background to account for possible clonal variation due to the different positions in the genome where genes targeted to delta sequences may have integrated. Our CRISPR-delta-integration method was successful in integrating the _T.r.eg2_ into _S. cerevisiae_ haploid and diploid strains. Significant differences in activity between different delta-targeted transformantsants, as well as between delta-targeted transformantsants and single locus-targeted transformantsants, were observed, as confirmed by _t_-tests (\(p\leq 0.05\)). At 72 h of cultivation, the best performing haploid M1744-\(\Delta\)-EG strain had an EG activity of 65 U/gDCW, while the best diploid MH1000-\(\Delta\)-EG strain had an activity of 110 U/gDCW. The lowest EG activity observed in the haploid M1744 strains was 15 U/gDCW, while the lowest EG activity observed in the diploid MH1000 strains was 36 U/gDCW. Based on these findings, the diploid MH1000 strain outperformed the haploid M1744 strain in terms of EG activity levels when the gene was targeted to the delta sequences. It was also clear that significant clonal variation was evident in these transformants. Interestingly, single gene targeting to Ch10 or Ch11 in the haploid M1744 background led to similar and even higher EG activities compared to the delta-targeted strains. However, in the diploid strain background, EG activities of 3- to 5-fold higher were observed for some of the delta-targeted transformantsants, compared to their Ch10 or Ch11 targeted counterparts. We used qPCR to determine the number of _T.r.eg2_ copies integrated into the delta sites (Table 3). M1744 strains that integrated _T.r.eg2_ at the delta-sequences only contained 1 copy, while the highest copy number observed among MH1000 strains tested was 3. \begin{table}
\begin{tabular}{c c} \hline _T.r.eg2_ **Transformed Stains** & _T.r.eg2_ **Copy Number** \\ \hline M1744-Ch10-EG2 (Haploid) & 1 \\ M1744-Ch11-EG2 & 1 \\ M1744-Ch12-EG2 & 3 \\ M1744-\(\Delta\)-EG2-1 & 1 \\ MH1000-Ch10-EG2 (Diploid) & 2 \\ MH1000-Ch11-EG2 & 2 \\ MH1000-Ch12-EG2 & N.D. \\ MH1000-\(\Delta\)-EG2-1 & 3 \\ MH1000-\(\Delta\)-EG2-1 & 1 \\ \hline _T.e.cb1t_ **transformed stains** & _T.e.cb1t_ **copy number** \\ \hline M1744-Ch10-CBH1 & 1 \\ M1744-Ch11-CBH1 & 1 \\ M1744-Ch12-CBH1 & 4 \\ M1744-\(\Delta\)-CBH1-1 & 2 \\ M1744-\(\Delta\)-CBH1-2 & 1 \\ M1744-\(\Delta\)-CBH1-3 & 2 \\ M1744-\(\Delta\)-CBH1-4 & 2 \\ \hline \end{tabular}
\end{table} 
Table 3: Copy numbers of heterologous cellulase genes integrated at different chromosomal sites in the various transformants. Numbers after the strain names indicate different transformants of the same strain that were tested and match the numbers indicated in Figures 3 and 4.