**4. Safety assessment and regulatory framework of GE systems**

### **4.1 Off-target effects**

One of the main limitation of GE systems is off-target effects, when nucleases, along with target regions, affect other parts of the genome [7–10, 39, 44]. It stems from the fact that the efficiency of DNA cleavage by nucleases depends both on nuclease activity and the availability of the target site and also the affinity of the DNA-binding domain. Moreover, the designed nucleases specificity largely depends on the binding affinity of the nuclease to DNA. In addition, FokI domain dimerization and interaction of Cas9 with PAM may also play an important role [17, 55, 56].

The preliminary comprehensive bioinformatics analysis to choose of specific sites for the introducing of DBS can minimize the off-target effects of the GE system. When choosing the desired sites, it should avoid regions with repeated sequences, as well as regions with high homology to other regions of the genome [17, 55, 56]. In addition, to minimize off-target effects and cellular toxicity of ZFN and TALEN heterodimerization of FokI nuclease is used [19, 21]. Another effective way to reduce the frequency of unintentional mutations is the use of the sgRNA/ aptazyme system (ligand-dependent ribozyme) [56].

### **4.2 Regulatory framework**

The legal and regulatory framework in regard to GE plants in different countries has a great impact on their competitiveness. In most countries, the current biosafety framework is meant to regulate transgenic GMOs. Currently, only Argentina and Brazil have adopted additional legislation for crops obtained using GE techniques [57].

There are two main approaches to defining the regulatory framework for GMOs in global legislation: one is process-based and the other is product-based. In the European Union, a standard is focused on the first approach, while in Canada – on the second. In the United States, a hybrid system is used: the decision on whether a crop belongs to GMOs is based on the first approach, and the risk assessment – on the second. In this case, GE cultures are considered individually [13]. Thus, the same GE culture can be classified in different ways depending on the regulatory framework. For example, in Argentina, crops that are classified as "zero segregants" are not regulated as GMOs [57]. In the EU, GE crops are subject to Directive 2001/18/EC and must undergo a full biosafety assessment procedure, as well as meet the requirements for GMO products [13, 57].

### **5. Future prospects**

GE tools are considered one of the most promising tools for practical agricultural biotechnology because of their high efficiency, relatively low cost, ease to use and multiplexing ability [7–10, 14]. Herewith, directed mutagenesis makes it possible to effectively "turn off" or silencing various genes, that helps to determine the functions of genes [17, 29, 34, 42, 43]. Multiplex targeting allow to reveal the role of individual genes and encoded proteins in intracellular signaling pathways and contributes to the engineering of multigenic agronomic traits in crops [7–10, 44].

Furthermore, CRISPR/Cas9 system may be used for spatial and temporal control of gene expression, as well as tissue-specific regulation of expression [42]. In the furtherance of this goal, influence on the *cis* regulatory elements of the gene promoter region has large potential and allows to change the expression level, expression of patterns, and tissue-specific expression [58, 59]. At the same time, BEs can be used to replace key nucleotides in *cis* elements to modulate the affinity of transcription factors and, accordingly, the level of expression [58]. Additionally, the introduction of mutations using programmed nucleases or BEs into the binding sites of the promoter regions will disrupt their interaction with the virulence proteins of pathogens and, thus, increase the resistance of crops [48, 58, 60].

Epigenomic studies are another promising uses of GE technologies. Herewith, the insertion of transposons into the promoter region due to the NHEJ or HDR mechanisms can affect the epigenetic status and, accordingly, the level of expression of commercially valuable genes [58]. It can be achieved through the using of such dCas9 systems as targeted DNA methylation and demethylation systems [58].

It should also be noted that an advanced uses of GE methods, in particular, CBE and ABE, is the introduction of single base changes into the microRNA (miRNA) binding site of the target gene without alteration of the amino acid sequence of the encoded protein. It will lead to disruption of miRNA/mRNA base pairing and, consequently, to disruption of mRNA cleavage due to the fact that the position and number of mismatches in the miRNA binding site strongly affect the efficiency of miRNA-mediated mRNA cleavage [58]. In turn, such disturbance of miRNA-mediated mRNA cleavage can be used to proper alignment of the target genes expression and, accordingly, can have effect on the development of many agronomical desired trait [58]. In addition, CRISPR/Cas system can be used for NHEJ- or HDR-mediated introduction of translation enhancers into the initiation codon of open reading frames, which provide fine tuning of gene expression at the translation level [58].

Thus, in summary it should be noted that GE systems, especially CRISPR/Cas and BEs, offer great opportunities for crop improvement.
