# An Overview of CRISPR-Based Technologies in Wine Yeasts to Improve Wine Flavor and Safety
## 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 [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 _archae_[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 [30]. During CRISPR adaptation (Figure 1a,b), 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 (Figure 1c), 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, Figure 1d) [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 [32]. These systems have created CRISPR gene editing, a technique that commonly utilizes the Cas9 gene [33]. Nowadays, Cas9 (CRISPR-associated protein 9, previously called Cas5, Csn1, or Css12), 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 [PERSON] and [PERSON] [34]. Therefore, CRISPR is the foundation of a prokaryotic adaptive immunity system [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 [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 (Figure 2). 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 [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 1 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 [38] (Figure 2). Figure 1: Schematic representation of the three stages of the CRISPR/Cas bacterial adaptive immune system: Injection of phage DNA into bacterial cell and integration into CRISPR sequence (**a**,**b**); CRISPR RNA biogenesis (**c**); Invader silencing (**d**). Adapted from [PERSON] and Severinov [31]. Figure 2: Schematic representation of CRISPR Cas9 _modus operandi_.