# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 1 Introduction
In the last two decades, due to the rapid progress in the field of yeast genomics, systems biology, and genetic engineering, new possibilities have emerged for creating wine yeast strains based on the knowledge of the molecular nature of the target trait or phenotype [16], resulting in the development of wine yeast strains with enhanced fermentation abilities, which, among others, provide the possibility of improving the sensory quality of the wine, increasing its pleasantness [17; 18]. The methods of genetic engineering, contrasting with the classical strain improvement, aim at changing a specific target _locus_ without affecting other sites and maintaining the remaining positive characteristics of the strains [16]. The genetic modification of _Saccharomyces cerevisiae_ wine strains has shown great potential in improving many enological traits, even if most of the studied strains have not gone out of the lab and have not been used in the industry [19]. Many of the works performed are involved with genes that modulate ethanol and glycerol content to generate wines with lower ethanol content [20] by overexpressing glycerol-phosphate dehydrogenase 1 and 2 (_GPD1_ and _GPD2_) genes [17]. Other works aim to expand the aroma-producing skills of wine yeast by overexpressing alcohol acetyltransferases 1 and 2 (_ATF1_ and _ATF2_) genes, involved in the synthesis of esters [21]. Nevertheless, only two GM wine yeast strains are officially registered and approved for commercial use [22; 23]. [PERSON] et al. [22] constructed a genetically stable industrial strain of _S. cerevisiae_ by integrating a linear cassette containing the _Schizosaccharomyces pombe_ malate permease gene (_mae1_) and the _Oenococcus eeni_ malodic gene (_mleA_) under control of the _S. cerevisiae PGKI_ promoter and terminator sequences into the _URA3 locus_ of industrial wine yeast. The ML01 yeast enjoys \"Generally Regarded as Safe\" (GRAS) status from the FDA and was the first genetically enhanced yeast to be commercialized. In the same year, [PERSON] and coworkers [23], by constitutively expressing the _DUR1,2_ gene in _S. cerevisiae_, were proficient in producing a yeast able to reduce the production of ethyl carbamate in wine by 89.1%. The yeast was genetically stable, and genotypic and phenotypic characterization and global gene expression patterns indicate that the genetically modified yeast strain was considered equivalent to the industrial parental strain. The metabolically engineered 522 EC- urea-degrading yeast is not considered transgenic since it does not contain foreign DNA or any antibiotic resistance marker genes. Thus, _Saccharomyces cerevisiae_ 522 EC- has received the GRAS status from the U.S. FDA. The genetic modification of yeast wine strains is, therefore, an emerging field of synthetic biology, which enables scientists to create yeasts with the exact phenotype desired for a given fermentation [24]. In recent years, CRISPR/Cas9 (_Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9_)-based genetic editing has become a mainstay in many laboratories, including manipulations done with yeast [25; 26]. This technique involves utilizing the natural \"adaptive immunity\" mechanism described in bacteria and _ archaea_ to develop a tool efficient for conducting precise genome editing of any organism. In this work, we aimed to overview the use of the CRISPR/Cas9 editing technique in wine yeasts to construct wine yeast able to increase flavor/aroma compounds in wine without the development of off-flavors and dangerous compounds to human health, aiding in the creation of \"safer wines.\"