# Molecular Tools to Exploit the Biotechnological Potential of _Brettanomyces bruxellensis_: A Review
## 3 CRISPR/Cas9 System in _B. bruxellensis_
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Procedure** & **Medium** & **OD\({}_{\mathbf{600 nm}}\) & \begin{tabular}{c} **Growth** \\ **Temperature** (\({}^{\circ}\)C) \\ \end{tabular} & \begin{tabular}{c} **Solution—Chemical** \\ **Compounds** \\ \end{tabular} & \begin{tabular}{c} **Transformation** \\ **Efficiency** \\ \end{tabular} & **References** \\ \hline LiAc/PEG & GYP; SCM & 0.60–0.75 & 28 \({}^{\circ}\)C & \begin{tabular}{c} M LiAc– \\ 50\% PEG \\ \end{tabular} & \begin{tabular}{c} 16 transformants \\ \(\mu\)g\({}^{-1}\) DNA \\ \(2.8\times 10^{3}\) \\ \end{tabular} & [49] \\ \multirow{2}{*}{\begin{tabular}{c} Electroporation \\ transformation \\ \end{tabular} } & GYP & 0.25–0.35 & 28 \({}^{\circ}\)C & \begin{tabular}{c} M LiAc– \\ 0.5—1 M sorbit \\ \end{tabular} & \begin{tabular}{c} 2.8 \(\times 10^{3}\) \\ transformations \(\mu\)g\({}^{-1}\) \\ DNA \\ \end{tabular} & [49; 56] \\ \multirow{2}{*}{\begin{tabular}{c} Spheroplast \\ transformation \\ \end{tabular} } & GYP; SCM & / & 28 \({}^{\circ}\)C & \begin{tabular}{c} Zymolyase–0.5 M–1 \\ M sorbitol \\ \end{tabular} & \begin{tabular}{c} DNA \\ plating efficiency \({}^{*}\) \\ \(75\%\); 7.4\% \\ \end{tabular} & [49] \\ \multirow{2}{*}{\
\begin{tabular}{c} Spheroplast \\ intraspecific fusion \\ \end{tabular} } & YPD; MM & 1.00 & 30 \({}^{\circ}\)C &
\begin{tabular}{c} Snail--PE buffer– \\ PEG 4000 \\ \end{tabular} & ** & [57] \\ \hline \hline \end{tabular}  
* Alternative method to evaluate the transformation efficiency. ** Data are not available.  
\end{table} 
Table 5: Available transformation approaches for _B. bruxellensis_ manipulation. in these species, delivering the ribonucleoprotein particle complex (RNPs) [71]. RNPs consist of a gRNA in complex with purified Cas9 protein. This complex is assembled in vitro and can be delivered using standard electroporation or transfection techniques [49; 70]. [PERSON] et al. obtained RNPs by co-incubating the crRNA designed to target ORF and trarcRNA. The authors achieved a targeted gene deletion of the _SSUI_ gene in the haploid strain AWRI2804, demonstrating growth inhibition of the mutants in the presence of SO2. Furthermore, they evaluated the possibility of using the same method in polyploid strains. They transformed the diploid strain AWRI1613 in order to delete both the _URA3_ genes, combining the _natMX_ cassette, containing long-flanking regions (1 kb) and RNPs. Two _\(\Delta\)URA3:__natMX_ mutants were obtained; their genome analysis revealed the integration of a single copy of the cassette and the concomitant deletion of the two different alleles, one for each mutant, thus showing the impossibility of deleting both alleles in a single step. Accordingly, a second deletion was attempted by transforming one of the mutants to combine the _kanMX_ cassette, which contained long-flanking regions (1 kb) and RNPs. The resulting _\(\Delta\)URA3:__kanMX_ showed the deletion of the second copy of the _URA3_ gene and the insertion of a single copy of the _kanMX_ cassette [66]. Therefore, these outcomes support the use of this method, which combines an expression-free CRISPR/Cas9 system and tailored _B. bruxellensis_ gene transformation cassettes and is potentially useful for polyploid strains in a stepwise transformation system.