# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 4 CRISPR System Applied to Wine Yeasts  
CRISPR/Cas9 genome-editing technology was first applied in _Saccharomyces cerevisiae_ in 2013, and the work was published in an article titled \"Genome engineering in _Saccharomyces cerevisiae_ using CRISPR-Cas systems\" by [PERSON] et al. [45]. There is an expanding interest in using CRISPR/Cas9 in _S. cerevisiae_ as one could infer from the increasing number of articles on PubMed. Using the keywords \"_S. cerevisiae_\" and \"CRISPR/Cas9,\" from 2014 to 2021, 220 articles were published (Figure 3A). The most recent was already published with the date of 2021, an article from [PERSON] and [PERSON] [46], titled \"CRISPR Nickase-Mediated Base Editing in Yeast\" where they describe the development of a CRISPR nickase system that can perform precise genome-wide base editing in _S. cerevisiae_, over five lab-working days, using a single Cas9 nickase and edit a broader genomic region by the avoidance of a double-strand break and subsequent nonhomologous end-joining. Again, and using the PubMed database, if the keywords \"wine yeast\" and \"CRISPR/Cas9,\" are used, only five articles appear [26; 47; 48; 49; 50], Figure 3B. Therefore, beginning with the oldest, from [PERSON] et al. [47], we verify that scientists from Toronto and Milan, in 2017, applied CRISPR to starter yeast strains to reduce urea production in Chardonnay and Cabernet Sauvignon grape musts. Urea is a precursor to ethyl carbamate (EC, urethane), a naturally occurring component of all fermented foods and beverages. Because EC has shown potential for carcinogenicity [51], the wine industry is interested in reducing EC levels in their products. EC can be produced during arginine metabolism by yeasts (Figure 4A,B) [51] or by lactic acid bacteria (_Lactobacillus_ and _Oenococcus_) [52]. This amino acid is usually abundant in grape juice (must) and is taken up by wine yeast as a nutrient. It enters the cell using arginine transporters-arginine permeases Can1, Alp1, and Gap1 [53; 54; 55]. When metabolized, it yields urea due to arginase activity [56]. If the urea cannot be metabolized, it accumulates inside the cell. Above a critical concentration, yeast cells release it into the wine during or at the end of fermentation. Urea can spontaneously react with the ethanol in wine to form EC, and the chemical reaction between urea and ethanol is exponentially enhanced at elevated temperatures. It is well studied that the formation of urea is affected by arginine metabolism, urea transport, and degradation [57], and arginine metabolism is easily altered by arginine transport in response to various nutrient conditions [58]; therefore, all these events also influence EC formation. Figure 4: _Cont_. Figure 3: (**A**) Number of articles on PubMed using the keywords “_S. cerevisiae_” and “CRISPR/Cas9,” from 2014 to 2021. Data retrieved from [[https://pubmed.ncbi.nlm.nih.gov/?term=S.+cerevisiae+CRISPR%2](https://pubmed.ncbi.nlm.nih.gov/?term=S.+cerevisiae+CRISPR%2) FCas9&sort=pubdate]([https://pubmed.ncbi.nlm.nih.gov/?term=S.+cerevisiae+CRISPR%2](https://pubmed.ncbi.nlm.nih.gov/?term=S.+cerevisiae+CRISPR%2) FCas9&sort=pubdate). (**B**) Number of articles on PubMed using the keywords “wine yeast” and “CRISPR/Cas9”. Data retrieved from [[https://pubmed.ncbi.nlm.nih.gov/?term=wine+yeasts+CRISPR%2](https://pubmed.ncbi.nlm.nih.gov/?term=wine+yeasts+CRISPR%2) Fcas9&filter=ssimearch1.fh4&sort=pubdate]([https://pubmed.ncbi.nlm.nih.gov/?term=wine+yeasts+CRISPR%2](https://pubmed.ncbi.nlm.nih.gov/?term=wine+yeasts+CRISPR%2) Fcas9&filter=ssimearch1.fh4&sort=pubdate). Accessed 22 November 2020. Wine yeast strains differ in their ability to rapidly catabolize urea during fermentation [56]. When excess urea accumulates in the cell's cytoplasm, it is released into its environment as must. High urea-producing yeasts are those that have a high capacity to degrade arginine to urea but a low urea-metabolizing ability [60].