## 4 Discussion
### Constructing Yeast Strains for Cellulose CBP  
Consolidated bioprocessing (CBP) is an improved process design to convert lignocellulosic feedstocks to biofuels in a single reactor using a single microbe or consortium [7; 42]. Heterologous expression of genes encoding various types of cellulases from fungi has been reported in recombinant _S. cerevisiae_[7; 8; 9; 10]. Constructing a CBP yeast strain requires the insertion of multiple genes into the genome of the host organism. This is often hampered by the availability of markers for subsequent rounds of engineering. Furthermore, for some applications, it is desired to have no DNA from bacterial origin remain in the industrial transformants. As CRISPR-Cas9 allows the insertion of a defined sequence without the need for leaving the selection marker in the transformant, it is an ideal technique for industrial strain development. This also allows the possibility of subsequent genetic modification in the same strain. We therefore set out to create diploid strains for CBP containing a core set of cellulase activities. It was previously reported that optimal synergy between cellulases relies on different ratios of the enzymes [29; 43]. The results shown in Figures 2-4 can thus inform the selection of loci for the integration of the cellulase genes. We observed that integrating _T.r.eg2_ and _T.e.cbh1_ to different loci yielded different levels of activity. These levels can thus be used to help optimize the ratio of the cellulases produced. As CBHI activity is the most essential for crystalline cellulose hydrolysis and delta sequence targeted _T.e.cbl1_ transformants yielded the highest CBHI activity, we targeted _T.e.cbl1_ to that locus. It was previously shown that EG activity, though essential for synergism, was not required at very high levels [29]. We, therefore, targeted _T.r.eg2_ to the Ch10 intergenic region but also created a version of the strains where this gene was targeted to the delta sequences for comparison. Finally, we attempted to target a BGL encoding gene (_A.a.bg1_) to the delta sequences but were unsuccessful, possibly showing that the limit for integrating to that target in our strains was reached. The _A.a.bg1_ was thus targeted to the Ch11 intergenic region. In summary, this yielded two strains with a core set of cellulases, namely MH1000-B11-E10-C\({}_{\Delta}\) and MH1000-B11-EC\({}_{\Delta}\) (Table 2). PCR amplification was used to confirm the presence of the four types of integrated genes in the CBP MH1000 yeast strains (Figure S1D). The ten-fold difference in CBH activity between these two strains (Figure 5B), despite the gene being targeted to the same genomic locus, may be indicative of gene copy number differences, varying loci of integration, or that there was a limitation in the number of different genes that could be efficiently targeted to the delta sequences in our strains. However, we have demonstrated that an industrial _S. cerevisiae_ strain could be successfully engineered with three cellulase-encoding genes without the need to maintain any selectable marker in the final strain. This makes the strain amenable to subsequent engineering steps. Furthermore, the observed differences in cellulase activities after integration at the different sites could be used to optimize the ratio of cellulases produced. Multiplex genome editing is another option to be considered as we performed the introduction of the four different genes into a single yeast strain over the course of several transformation rounds. Creating suitable gRNAs and an appropriate vector to express them is critical in achieving multiplex integration and minimizing transformation rounds. [15,44]. Despite the significant advancements in the development of numerous assisting tools for designing gRNAs for various Cas proteins [45, 46, 47, 48], the effectiveness of these gRNAs in vivo still has to be evaluated, which is even more critical for multiplex integration [49]. Additionally, the target locus selection has a substantial effect on the integration efficiency.