## 5 Conclusions  
This study aimed to use CRISPR-Cas9 to integrate multiple genes at different genomic locations with the goal of creating markerless CBP-enabled yeast strains. Our CRISPR-Cas9 systems enabled efficient gene integration in different chromosomal positions within the yeast genome. However, we observed significant differences in activity at different loci: (i) when the same gene (_T.r.eg2_) was integrated and (ii) when a different gene was integrated (_T.e.cbl1_). Differences in gene copy number were also observed between haploid and diploid yeast strains. While we showed that targeting genes to our selected Ch12 site yielded transformants with higher levels of EG and CBH activity than all other single-copy integration sites, we were unable to confirm that the gene was integrated at the selected position, casting doubt over the usefulness of this particular locus for strain construction due to off-target integration. The repeated delta sequences were targeted for multi-copy integration, and we discovered that delta integration frequently resulted in transformants with only one gene copy in haploid strains and two copies in diploid strains for both genes tested. _T.e.cbl1_ transformants where the gene was targeted to delta sites yielded higher activity than transformants that were targeted for single-copy integration. We also showed that while targeting _T.e.cbl1_ to the delta sites yielded transformants with the highest CBH activity, _T.r.eg2_ transformantsands had slightly lower EG activity when targetingthe delta sites in the haploid strain background. Our results clearly showed that there were gene-specific and locus-specific factors involved in obtaining high levels of heterologous protein production and that these factors cannot necessarily be predicted prior to testing the various loci and heterologous genes of interest. Applying this knowledge, we created CBP-enabled yeast strains based on an industrial diploid yeast strain, transformed in different rounds of transformation to create a rudimentary cellulase system. Three cellulase-encoding genes could be transformed into the yeast strain in a marker-free strategy making additional genetic manipulation possible. Furthermore, the direct conversion of crystalline cellulose to monomeric sugars without the addition of exogenous enzymes could be shown. It was observed that targeting most of the genes to the delta sequences had detrimental effects on the individual cellulase activities and on cellulose conversion. It may therefore be prudent to identify genomic loci that allow high-level production of enzymes on interest and create transformants with combinations of optimally targeted genes rather than relying on delta integration alone. Based on these findings, we concluded that our system was useful and easily applied in the metabolic engineering of _S. cerevisiae_ for biofuel production. It is likely also applicable to various wild-type and industrial strain isolates. While our rudimentary CBP strains were comparable to similar strains that were previously described (as reviewed in [PERSON] et al., [12]; they did not degrade most of the available cellulose in the cellulose conversion assay. These strains could be further improved by using strain engineering approaches or applying the CRISPR-Cas9 approach to more process-amenable yeast strains [54,55]. The markerless nature of the CRISPR-Cas9 methodology enables a vast amount of additional engineering to improve these strains. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app122312317/s1](https://www.mdpi.com/article/10.3390/app122312317/s1)]([https://www.mdpi.com/article/10.3390/app122312317/s1](https://www.mdpi.com/article/10.3390/app122312317/s1)): Table S1: Primers used in the study; Table S2: Gene integration target sites on different chromosomes; Figure S1: Confirmation of the cellulase genes integrated into haploid and diploid _S. cerevisiae_ isolates. Conceptualization, R.d.H. and O.J. ; methodology, R.d.H., G.R.v.L. and O.J.