**3.2 Using of GE approaches in plant breeding**

Nowadays, GE technologies are effectively used to create new varieties of agricultural crops with improved traits, such as increased yield, product quality and resistance to biotic and abiotic stresses. Such traits improvement is often carried out by introducing target mutations into the corresponding regulatory genes that control the development of undesirable traits leading to the suppression of their activity [7–10].

In this section, we review key advances in crop trait enhancement using GE techniques and discuss their prospects for improving food security.

### *3.2.1 Crop yields increase*

Productivity is one of the most important economically valuable traits of agricultural crops. At the same time, this trait is also one of the most difficult to improve by conventional breeding methods [44]. It's explained by the fact that yield is often a quantitative multigenic trait, the development of which is controlled by multiple quantitative trait loci (QTL) [45]. Additionally, traditional yield-based selection is complicated by QTL introgression between different varieties, which is especially pronounced in the case of closely linked loci [44, 45].

In this regard, GE technologies represent a promising tool for the rapid and directed mutagenesis of target genes [7–10, 13]. Herewith, the most effective way to increase yields using genome editing technologies is to knock out ("turn off") genes negatively affecting the yield [44]. For example, CRISPR/Cas9-based "turn off" of

the functions of yield negative regulators (*Gnla*, *DEP1* and *GS3*) in rice has led to yield improvement, that manifested itself as increased number of grains in panicles and a larger grain size, respectively. It should also be noted that this gene knockout is inherited and observed at least in the T2 generation inclusive [10, 44].

Additionally, there is evidence that CRISPR/Cas9-based multiplex knockout of the main negative regulators of rice grain weight (*GW2*, *GW5*, and *TGW6*) allowed to significantly increase the weight of grains. Similar results were obtained by CRISPR/Cas9-mediated knockout of the *GASR7* gene (a negative regulator of the wheat grain width and weight). In addition, CRISPR/Cas9-based silencing of *OsGn1a*, *OsDEP1*, *OsGW2*, *OsGW5*, *OsTGW6*, *OsGS3*, *OsIPA1*, *OsPYLs*, *OsCCD7*, *OsLAZY1* and *NtPDR6* genes in wheat allowed to improve the yield-related characteristics [44, 46].

Also, it was shown that the CRISPR/Cas9-xyr5APOBEC1-mediated single base mutations in two rice genes, *NRT1.1B* and *SLR1* improved the efficiency of nitrogen utilization and increased yield [39]. Also, CRISPR/Cas9-mediated knockout of genes contributing to yield improvement allows to amend this economically valuable trait in many other crops [44, 46].

#### *3.2.2 Product quality improving*

Products quality is another economically valuable trait, the selection of which by traditional methods is accompanied by significant difficulties. Thereat, selection for this trait is complicated both by the difficulty in obtaining targeted mutations by the methods of chemical and physical mutagenesis, and the presence of negative correlations between the traits of quality and yield [11]. GE technologies allow to cope with the deficiencies of chemical and physical mutagenesis due to the ability to introduce targeted mutations into the genome and improve the nutritional properties of crops [7–10, 44].

Let us consider some examples of the potential application of GE methods for modifying the chemical composition of plants. For example, silencing one of the key genes of phytate biosynthesis *ZmlPK* by TALEN and CRISPR/Cas9 systems allowed to reduce its content in corn (*Zea mays*) [10, 44]. Herewith, the feed value of such corn grain is much higher due to the fact that phytate is considered an anti-nutritional element, reducing availability for digestion of proteins and minerals. Similar results were obtained in barley with TALEN-mediated knockout of the *HvPAphy* gene, which plays an important role in phytate biosynthesis [10, 44].

Also, TALEN-based "turn off" of *VInv* gene encoding vacuolar invertase allows to obtain potatoes (*Solanum tuberosum*) without the potential carcinogen acrylamide, which is formed during frying as a result of the reaction between reducing sugars and free amino acids [10, 44]. Additionally, TALEN system was used to knock out the *OsBADH2* gene in rice that resulted to an increase in 2-acetyl-1-pyrroline [44], which is responsible for the smell of cooked rice. Along with this, TALEN-mediated mutagenesis of the *FAD2-1A/B* gene in soybeans increased the content of oleic acid [44].

CRISPR/Cas9-based targeting of conserved regions in the α-gliadin genes has allowed to create wheat lines with reduced gluten immunoreactivity [44]. At the same time, CRISPR/Cas9-mediated multiplex mutagenesis of *SGR1*, *LCY-E*, *LCY-B1*, *LCY-B2* and *Blc* genes involved in lycopene biosynthesis contributed to the production of tomato lines with an increased content of lycopene [9, 10].

In addition, it was reported that CRISPR/Cas9-mediated knockout of genes responsible for amylose biosynthesis: *GBSS* gene in potatoes and the *Wx1* gene in maize allowed to obtain potato and maize lines with a reduced amylose content [9, 10, 44]. The opposite result was obtained in rice by silencing of the *SBEI* and

*SBEIIb* genes responsible for the biosynthesis of starch [44]. CRISPR/Cas9 system was also used to decrease the level of linolenic acid and to increase the level of oleic acid in *Camelina sativa* by multiplex knockout of *FAD2* homeologues [46].
