## 4 Discussion  
### Endoglucane Integration and Activity  
[PERSON] et al. [17] previously reported that expression cassettes might integrate elsewhere in the genome via break-induced replication (BIR), resulting in strains where the gene of interest is difficult to localize. This is a known side-effect of some protospacer sequences in CRISPR applications. Despite using the E-CRISP predictive tool to avoid this problem, it still occurred for the Ch12 targeting site, though not for targeting the Ch10 or Ch11 sites. We observed, in agreement with previous reports, that the DSB created by the guide RNA-targeted Cas9 endonuclease was critical for correct integration at a significantly higher efficiency than endogenous homologous recombination alone [16].  
As a diploid strain, MH1000 may be expected to offer more potential sites for _T.r.eg2_ integration than the haploid M1744. The copy number data (Table 3) was thus in agreement with the observed higher levels of activity for the MH1000-\(\Delta\)-EG2-1 strain compared to its single integration counterparts in MH1000 and the delta-targeted M1744 transformants (Figures 2 and 3). However, the different levels of activity of the single-copy transformants and the various delta-targeted transformants show that the locus of integration also had a significant effect on activity.  
Numerous researchers have demonstrated that multi-copy integration via the delta sequences is an effective method for producing recombinant proteins, demonstrating increased expression levels using this method [35]. Although the number of integrated gene copies produced by delta-integration varies significantly according to its length, integration of 1 to 80 copies have been reported [36]. [PERSON] et al. [37] integrated 40 copies of a 3.5 kb donor DNA sequence into the delta sequence using the CRISPR-Cas9 system of _S. thermophilus_. [PERSON] et al. [38] also reported the breakdown of the delta sequence by the CRISPR system and subsequent delta integration. The highest copy numbers obtained  
Figure 5: Enzyme activity profiles of recombinant yeast strains after 72 h cultivation. (**a**) The activity of EG2 producing diploid strains on CMC. The untransformed MH1000 strain was used as a negative control. (**b**) Enzyme activity profiles of CBH-producing strains on MU-Lac. (**c**) Enzyme activity profiles of BGL-producing strains on pNP-G. (**d**) Cellulose conversion of the strains measured on Avciel. In (**d**), bars with lines represent values measured after 24 h of the conversion assay, and bars with blocks represent 48-h values. All values obtained were normalized using the dry cell weight (DCW) of each strain at 72 h of incubation. All values represent mean values of assays conducted in triplicate with error bars indicating standard deviation.  
with the 8, 16, and 24 kb donor DNAs were 11, 10, and 18, respectively. In our study, we were only able to obtain 1-3 copies with our \(\sim\)2 kb donor DNA. These differences may stem from differences in the strain backgrounds, differences in donor DNA length or the breakdown efficiency of our chosen delta-sequences by CRISPR-Cas9 [35] and/or chromosomal rearrangements, influenced by stressful environmental conditions [39].