The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e., the insertion of them into a target genome region, are between one and two orders of magnitude less frequent. First and foremost, this is associated with the specific features of the repair systems of higher eukaryotes and the availability of the donor template in accessible proximity during double-strand break (DSB) repair. This review briefs the main repair pathways in plants according to the aspect of their involvement in genome editing. The main methods for increasing the frequency of knock-ins are summarized both along the homologous recombination pathway and non-homologous end joining, which can be used for plant objects.

Two lines of transgenic carrot plants producingMycobacterium tuberculosisproteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.002% of TSP for CFP10. As has been shown, oral immunization of mice induces both the cell-mediated and humoral immunities. These data suggest that the proteins in question are appropriate as a candidate edible vaccine against tuberculosis.

Accurate recognition of regulatory elements in promoters is an essential prerequisite for understanding the mechanisms of gene regulation at the level of transcription. Composite regulatory elements represent a particular type of such transcriptional regulatory elements consisting of pairs of individual DNA motifs. In contrast to the present approach, most available recognition techniques are based purely on statistical evaluation of the occurrence of single motifs. Such methods are limited in application, since the accuracy of recognition is greatly dependent on the size and quality of the sequence dataset. Methods that exploit available knowledge and have broad applicability are evidently needed.

The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria. The role of protein targeting to certain cell organelles depending on specific features of recombinant proteins and the effect of this targeting on the protein yield are discussed, in addition to the main directions of the search for signal sequences based on their primary structure. This knowledge makes it possible not only to predict a protein localization in the cell but also to reveal the most efficient sequences with potential biotechnological utility.

The development and improvement of molecular biology methods have led to the creation of new technologies that make it possible to modify plant genomes by transferring and integrating into the genomes’ heterologous genes from various expression systems (genetic engineering), as well as inducing knockouts of one or more target genes of interest (genomic editing). The development of genome-editing methods is a new milestone in the development of modern breeding methods and certainly relies on the knowledge and technologies developed for transgenesis. This review will discuss issues related to the advantages and disadvantages of both technologies for improving the economically valuable traits of important crops.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.