Wine aroma and flavor are the major attributes determining the preferences of wine consumers [1]. When drinking wine, in the mouth, the wine warms, making aromatic molecules escape up the retronasal passage to reach the olfactory area, activating receptors in the olfactory epithelium [2]. At the same time, the taste buds, about 10,000 of them distributed all over the tongue and in the inside of the mouth, sensitive to the six basic “tastes”—sourness or acidity, sweetness, bitterness, saltiness, the “savory” umami and the “fat-taste”—are excited [3]. Wine also contains components that can influence the inside of the mouth: (i) tannins that taste bitter and let as perceived astringency; (ii) alcohol, which affects our nervous system, in wines that are particularly alcoholic, acidic, and with a high concentration of phenolics, can leave a “hot” sensation on the palate after tasting; and (iii) gassy carbon dioxide, which has a physical, tactile effect, varying from a gentle prickle to an uncomfortably overwhelming froth, which, due to its perceived acidity, also makes the wine taste fresher [4]. At the same time, a sensory experience, which we call “flavor,” occurs, and it can be described as “(…) a mingled experience based on human judgment, built on personal differences in perception thresholds” [5].

Yeasts possess a major role in wine chemical composition, not only because they produce enzymes that convert neutral grape metabolites into flavor active compounds but also because they produce secondary metabolites during fermentation and can extract flavor components from grape solids and by cell autolytic degradation [6]. These reactions vary with the yeast species and strains contributing to the fermentation. Therefore, one of the tools that can assist winemakers to produce wines with specific flavor profiles is the choice of the yeast or yeast culture strains to conduct fermentation [5]. The “prima donna” yeast will be the one that efficiently completes the conversion of grape sugars to ethanol, carbon dioxide, and sensorially important metabolites, without the development of off-flavors [7]. Another important characteristic consists of the modification of grape-derived constituents (glyco- and cysteine-conjugates), which will enhance the wines’ varietal character [7].

Moreover, the production of a “safer wine” is also one of the concerns in the wine industry. Winemakers feel the need to use yeast strains with peculiar features, such as low production of undesirable metabolites and already named “harmful compounds” such as SO₂, acetaldehyde, biogenic amines (BAs), and ethyl carbamate (EC), among others ^[8][9][8,9].

Beginning in the 1990s, classical strain improvement methods focused on successive stages of mutagenesis and selection [10] have been used to obtain yeast starters for winemaking. These methods are extensive and time-consuming, requiring the screening of a significant number of isolates [11]. However, an evolution occurred, and strains’ optimization, aiming to achieve mutants with the desired phenotype, by serial or the continuous culturing of a particular yeast for many generations under a selective pressure environment, began to be the more promising technique [12]. The supremacy of this last approach concerning the optimization of wine yeast is characterized by the creation of strains with increased sulfite tolerance and glycerol accumulation [13], strains with improved resistance to acetic acid and even acetic acid consumption [14], as well as enhanced viability and antioxidant production [15], among other interesting enological traits.

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][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].

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 ^[20][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 ^[21][22][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.

2. What Is CRISPR and How Does It Work?

It is known that prokaryotes have evolved a variety of innate defense mechanisms to be able to survive the constant attack by deadly viruses or other invading mobile genetic elements ^[23][27]. One of their weapons is the clustered and regularly interspaced short palindromic repeats, also known as CRISPR, a family of DNA sequences found in the genomes of bacteria and archaea ^[24][25][28,29]. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote cells. Therefore, the CRISPR-Cas system is an immune system that confers prokaryotic cells’ resistance to external genetic elements, present within plasmids and bacteriophages ^[26][30]. During CRISPR adaptation, the injection of phage DNA into bacterial cells activates the Cas1-Cas2 adaptation module proteins, which excise spacer-sized fragments of phage DNA and channels them for incorporation into the CRISPR array. During CRISPR RNA biogenesis, the CRISPR array is transcribed, and the resulting pre-crRNA is processed at repeat sequences to generated crRNAs. Individual crRNAs are bound by Cas protein effectors. When phage DNA with sequences matching a CRISPR spacer appears in the cell, effectors programmed by appropriate crRNA bind to it, and the resulting R-loop complex is destroyed by Cas executor nuclease (foreign DNA degradation or invader silencing) ^[27][31]. RNA holding the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA and RNA-guided Cas proteins cut foreign RNA ^[28][32]. These systems have created CRISPR gene editing, a technique that commonly utilizes the Cas9 gene ^[29][33]. Nowadays, Cas9 (CRISPR-associated protein 9, previously called Cas5, Csn1, or Csx12), a 160 kilodalton protein, is greatly utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell’s genome. The CRISPR-Cas9 genome-editing technique discovery was a significant contributor to the Nobel Prize in Chemistry in 2020, being awarded to Emmanuelle Charpentier and Jennifer Doudna ^[30][34]. Therefore, CRISPR is the foundation of a prokaryotic adaptive immunity system ^[31][35] and Cas9 is an endonuclease that makes double-strand cuts upstream to a protospacer adjacent motif (PAM) sequence in the target genome. Cas9 is driven to its target locus by a small programmable RNA—an RNA sequence that can be engineered to a specific target ^[32][36]. Streptococcus pyogenes Cas9 recognizes the 3-nucleotide PAM site, NGG (where N is any nucleotide, followed by two guanines (G)), and cleaves between the third and fourth nucleotides 5’ to the PAM site. CRISPR and homologous regions complement one another and generate precise, flawless, and marker-free genome edits. CRISPR technology also allows multiplex genome editing once Cas9 can interact with different guide RNAs, allowing the attack of various targets in a single cell ^[33][37]. The RNA binds to a 20-base pair complementary sequence in the genomic DNA. Guide RNA activity derives from a duplex of two RNA molecules: a CRISPR RNA (crRNA), which is complementary to the target, and a transactivating CRISPR RNA (tracrRNA). The crRNA and tracrRNA form a duplex since they possess a small homologous region. Association of the Cas9 with the crRNA|trRNA duplex molecule triggers a conformational change in the former (Cas9), letting it bind DNA at the adjacent PAM site and implement the double-strand cut. Consequently, both complementarities to the crRNA and an adjacent PAM site are required for targeting Cas9-mediated cleavage ^[34][38].

3. CRISPR in the Vineyard

There are several methods for giving grapevine more favorable properties. The commonly used and widely accepted form of human control is conventional plant breeding by crossing two plants. Sexual propagation combines the DNA and the properties of the plant. In vineyards, it has occurred naturally for many millenniums or by the action of humans in recent centuries ^[35][39]. To avoid losing already present favorable characteristics in the wine grape due to the cross, like adaptations to climate changes ^[36][40], grapevines are crossed back with the original plant. After a long process of selection (for the right properties), backcrossing, selection, backcrossing, and so on, eventually, a grapevine is produced that is like the original plant but with the additional required property. Genetic modification by cutting and pasting DNA from one plant to another, using vectors such as Agrobacterium tumefaciens and plasmids, is another well-developed technique. A new technique is gene editing using the CRISPR/Cas9, a method that allows adjusting the plant’s DNA. By back-crossing, it is possible to remove the plant-foreign DNA ^[37][41]. Grapes are vulnerable to pests and environmental stresses due to a lack of genetic diversity ^[38][42]. One example is the crisis created by the phylloxera bug in the late nineteenth century that devastated vineyards across Europe. Therefore, scientists are trying to keep the flavors and aromas of grapes while protecting them against environmental stresses. Researchers are experimenting with CRISPR, and one of the first works was performed by Ren and coworkers ^[37][41] in the grape variety Chardonnay. The former authors transformed “Chardonnay” embryogenic cell masses (calli) to gain point mutations in the L-idonate dehydrogenase gene. They were able to regenerate plants with altered production of tartaric acid and vitamin C. Moreover, using two sgRNAs targeting distinct sites in the gene, they obtained a 100% mutation frequency in the transgenic cell mass. In another study, Wang et al. ^[39][43] utilized the CRISPR/Cas9 system in the cultivar “Thompson Seedless” to show that the transcription factor gene VvWRKY52 is necessary for vulnerability to Botrytis cinerea. Recently, Li and coworkers ^[40][44] used the CRISPR/Cas9 system to study the role of grapevine PR4 in the resistance to downy mildew and demonstrated that the CRISPR/Cas9 system can be a high-efficiency tool for generating targeted mutations in grapevine and is an excellent technology for gene functional analyses in grapevine. However, what about in wine yeasts?

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 DiCarlo et al. ^[41][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. The most recent was already published with the date of 2021, an article from Kuroda and Ueda ^[42][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 ^{[22][43][44][45][46]}[26,47,48,49,50]. Therefore, beginning with the oldest, from Vigentini et al. ^[43][47], itwe is verifiedverify 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 ^[47][51], the wine industry is interested in reducing EC levels in their products. EC can be produced during arginine metabolism by yeasts ^[47][51] or by lactic acid bacteria (Lactobacillus and Oenococcus) ^[48][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 ^[49][50][51][53,54,55]. When metabolized, it yields urea due to arginase activity ^[52][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 ^[53][57], and arginine metabolism is easily altered by arginine transport in response to various nutrient conditions ^[54][58]; therefore, all these events also influence EC formation. Wine yeast strains differ in their ability to rapidly catabolize urea during fermentation ^[52][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 ^[55][60]. Low urea-metabolizing ability may result from the low activity of urea amidolyase, inhibition of amidolyase activity by the presence of high levels of ammonia, deficiencies of cofactors required by amidolyase, or low activity due to hyperactive arginase ^[55][60]. Genetic as well as environmental factors influence the amount of urea released by the cells. There are, already, some commercial yeast strains, Lallemand 71B^®, Red Star SC1120^®, and Premier Cuvée (PdM)^®, described as producing low levels of urea ^[56][61]. 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 ^[57][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 ^[58][59][92,93].