# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 3 CRISPR/Cas9 System in _B. bruxellensis_  
Genome editing using the CRISPR/Cas9 system is a valuable instrument for yeast genome manipulation that allows researchers to study the biochemical pathways or change the phenotypic characteristics of strains using metabolic engineering [47; 55]. This breakthrough technique enables the creation of site-specific mutations in targeted locations and the integration of multiple DNA constructs in a single transformation event [59; 60; 61; 62]. The great advantage of the CRISPR/Cas9 system is the use of a single Cas9 nuclease that can modify multiple alleles/genes simultaneously [62]. From this perspective, this method appears ideal for use in species such as _B. bruxellensis_ that present different ploidy states [63]. Although the CRISPR/Cas9 system approach has been widely studied and applied in _S. cerevisiae_ strains, its use in other yeast species is still challenging [64; 65]. Several strategies have been developed for non-_Saccharomyces_ yeasts (_Yarrowia lipolytica_, _Pichia pastoris_ syn. _Komagataella phaffi_, _Kluyveromyces lactis_, _Candida albicans_, and _C. glabrata_), but the reported efficiencies are still lower than in _S. cerevisiae_ strains [55]. Indeed, the crucial point of the CRISPR/Cas9 system is the insertion of double-strand breaks (DSB) and the subsequent recombination strategy; in fact, nonhomologous end joining (NHE) is the main route for most yeasts other than _S. cerevisiae_, which applies the homologous recombination (HR) strategy [65]. These two phenomena occur with different efficiencies: HR achieves precise mutations or alterations in the target locus, whereas NHE) results in genetic mutations, deletions, and translocations [66]. Regrettably, NHE is the preferred DNA-repair approach in _B. bruxellensis_, which may prevent the possibility of overcoming the translation or deletion of genes induced by CRISPR/Cas9, limiting the survival of the transformed clones [66]. An interesting method was proposed by [PERSON] et al. [67] in 2017, in which the NHE]-imped _P. pastoris_ CBS 7435 mutant \(\Delta\)ku70 and the CRISPR/Cas9 system were combined to perform transformation experiments, increasing the HR efficiency. This strategy allowed them to obtain a CRISPR/Cas9-mediated integration of markerless donor cassettes with an efficiency of 100%, and, even though the transformation efficacy was lower than that achieved using conventional strategies, all the transformants demonstrated correct integration [67]. Subsequently, in 2020, [PERSON] et al. described the application of the CRISPR/Cas9 system in _B. bruxellensis_[66]. In that study, several experiments were performed to obtain an expression-free CRISPR/Cas9 system to promote HR-based gene deletion in combination with _B. bruxellensis_ gene transformation cassettes (described in the work of [PERSON] et al. in 2018 [54] and listed above) for the deletion of the _SSUI_ gene responsible for sulfite tolerance [68; 69; 70]. This method was previously investigated for pathogenic _Candida_ species (other than _C. albicans_) by [PERSON] et al. (2017). These authors bypassed the inefficient heterologous expression of the CRISPR/Cas9 system components