### Tocopherol Synthesis
#### 2.1.3 Genetic Approaches for Boosting Isoprenoid Production in Plants
Silencing of the _CCD4_ gene using CRISPR/Cas9 in banana plants resulted in an increase of \(\beta\)-carotene content of 1.3-1.4 times in leaves and of 2.3-2.7 times in roots compared to the wild-type plants [144]. A new step in plant biotechnology involved the production of marker-free rice plants with an increased content of carotenoids in grains by the insertion of a biosynthesis gene cassette into two specially selected regions of the plant genome, called genomic safe harbors (GSHs). The integration of genetic constructs into GSHs avoids negative consequences of accidental random insertions into vital regions of the plant genome. The carotenoid biosynthesis gene expression cassette included the carotenoid desaturase gene from _Ervinia__uredvora_ (SSU-crtl) and _maize phytoene synthase_ (_ZmPsy_) under the control of the endosperm-specific glutelin promoter [145]. The resulting rice plants accumulated significant amounts of \(\beta\)-carotene in the endosperm compared to non-transgenic plants without \(\beta\)-carotene in the endosperm. Metabolic engineering, complemented by genome editing techniques, allows researchers to improve desired experimental results. Thus, the transfer of a transgenic construct including an expression cassette of carotenoid biosynthesis genes (maize _PSY_, Arabidopsis _ORHis_, barley _HGGT_), together with CRISPR/Cas9 sequences to knock out the \(\beta\)-_carotene hydroxylase 2_ (_BCH2_) gene, increased the carotenoid content in Arabidopsis seeds by 5.3 times and their stability during ripening and storage. Due to the knockout of the _BCH2_ gene, the negative effect of increased carotenoid content on seed storage and germination was reduced, since the pool of hydroxylated \(\beta\)-carotene, which is a precursor in abscisic acid biosynthesis, decreased [146]. We summarized the results of genome editing by CRISPR/Cas9 in various species of higher plants indicating target genes and the type of editing in Table 2.  
\begin{table}
**Proteins** & **Species** & **Target Genes** & **Anti-Oxidants** & **Editing Type** & **Result** \\ \hline Kaempferol 3-O- & & & & & UQ content in the double knockout \\ rhamnosyltransferase & _A. thaliana_ & _At1g30530, At5g17050_ & UQ & Knockout as a result of deletion and insertion & double knockout \\ and kaempfer & _A. thaliana_ & _At5g17050_ & UQ & Deletion and insertion & represented 160\% of wild-type level [147] \\ 3-O- & & & & & \\ glucosyltransferase & & & & & \\ \hline \hline \end{tabular} \begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{\multirow{4}{*}{PSY, phytoene synthase}} & \multirow{4}{*}{_Oryza sativa_} & \multirow{4}{*}{_ZmPsy_} & \multirow{4}{*}{Carotenoids} & \multirow{4}{*}{ \begin{tabular}{c} _Marker-free targeted_ \\ insertion at \\ pre-determined plant \\ genomic safe harbors \\ \end{tabular}  } & \multirow{4}{*}{\ \begin{tabular}{c} _Marker-free targeted_ \\ genomic safe harbors \\ \end{tabular}  } & \multirow{4}{*}{ \begin{tabular}{c} _Marker-free targeted_ \\ genomic safe harbors \\ \end{tabular}  } \\ PSY, & & & & \\ phytoene synthase & _Oryza sativa_ & _ZmPsy_ & Carotenoids & _turedvora carotenoid_ \\ \cline{3-4} \  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Proteins** & **Species** & **Target Genes** & **Anti-Oxidants** & **Editing Type** & **Result** \\ \hline _LCY-E_, lycopene & & _DQ100158_ & & \\ \(\varepsilon\)-cyclase; & & (_SGR1_), & & \\ _Blc_, beta-lycopene & & _EL533951_ & & \\ cyclase; & & (_LCY-E_), & & \\ _LCY-B1_, lycopene & _S. lycopersycum_ & _XM_, _010313794_ & Knockout as a result of deletions, insertion, substitution & Lycopene content in tomato fruit was increased about 5.1-fold [132] \\ _LCY-B2_, lycopene & & (_LCY-B1_), & & \\ _SGR1_, Stay-green 1 & & (_LCY-B2_) & & \\ \hline _O.