# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 4 CRISPR System Applied to Wine Yeasts
Aiming to increase the knowledge about the _S. cerevisiae_ genes affecting the production of phenylethyl acetate, [PERSON] and coworkers, in 2017 [76], investigated the polygenic basis of 2-phenylethyll acetate production, using two random segregants (BTC.1D and ER18) derived from two nonselected diploid strains as parents and identified offspring with high 2-phenylethyll acetate production. Several major QTLs were mapped and two novel causative alleles of _TOR1_ (a gene involved in nitrogen regulation of growth) and _FAS2_ (a gene that encodes a subunit of the fatty acid synthetase complex) were identified as superior alleles derived from strain BTC.1D. Exchange of the superior alleles of TOR1 and FAS2 by CRISPR/Cas9-mediated allele exchange into the ER18 parent strain increased by 70% the 2-phenylethyll acetate production [76]. CRISPR genome-editing techniques have also been applied in many nonconventional yeasts, such as _Candida_ species: in _Candida tropicalis_, for the biosynthesis of \(\beta\)-carotene and its derivatives [77] and _Candida glycerinogenes_ for the coproduction of ethanol and xylonic acid [78]; in _Pichia pastoris_ (syn. _Komagaatella phaffii_), to improve its efficiency for the production of high-value pharmaceuticals [79]; in _Ogataen polymorpha_ (syn. _Hansenula polymorpha_), a methylotrophic yeast important for the production of bioethanol because it can grow at high temperatures (up to 50 \({}^{\circ}\)C) and assimilates several carbon sources including glucose, xylose, and cellobiose [80]; in the xylose, catabolizing yeast _Scheffersomyces stipitis_[81]; in _Issatchenkia orientalis_, aiming to develop a strain highly efficient in the for the production of biofuels and chemicals [82], in _Kluyveromyces lactis_[83] and, _Kluyveromyces marxianus_[84], in _Schizosaccharomyces pombe_[85], and in _Yarrovia lipolytica_ useful as a biomanufacturing platform for a range of industrial applications, including renewable chemicals and enzymes for fuel, feed, olechemical, nutraceuticalal and pharmaceutical applications [86]. Important work was developed by [PERSON] and coworkers [50] in _Brettanomyces bruxellesensis_. This yeast is known for its role in the spoilage of fermented beverages including wine, beer, and cider [87]. However, _B. bruxellensis_ has several characteristics important for biotechnological applications, such as being able to utilize nitrate as a nitrogen source [88], having tolerance to high levels of ethanol and low pH [89], and being able to assimilate cellobiose, besides other usual carbo sources [90]. [PERSON] and coworkers [50] aimed to facilitate targeted genome editing in _B. bruxellensis_, by combining optimized selection cassettes with various lengths of flanking DNA and an expression-free CRISPR-Cas9 system, assessing, in this way, the ability to promote homologous recombination-based gene deletion. They were able to delete _SSUI_ genes (conferring sulfite tolerance) in the AWRI2804 _B. bruxellensis_ strain. However, while the use of CRISPR for gene editing is regarded to be outside the scope of definitions for genetic engineering or genetically modified organisms (GMOs) labeling in the United States and Japan, as long as the modification occurs in nature or could be achieved by traditional breeding, the Court of Justice of the European Union ruled, in 2018, that any organism made with in vitro mutagenesis (such as CRISPR-Cas) falls within the definition of GMO [91]. There are attempts by the scientific community to lead a reconsideration of the decision in the European Union (EU), which are obsolete in light of the nature of gene-editing techniques [92; 93].