Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants

Abstract

Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic “safe harbors”) using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of “euchromatization” of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

“…Therefore, the risk of interference with the coordinated expression of own genes is lower after insertion of a transgene; accordingly, the selection of a proper insertion locus providing the most active protein synthesis remains the main task. On the one hand, housekeeping genes are most suitable for this purpose because their activity is maintained at a high level during the entire cell lifespan [ 121 ]. In particular, the delivery of a target gene called dIFN , coding for human γ-interferon, to the transcriptionally active region of the histone H3.3 gene with the help of CRISPR/Cas9 has helped to obtain monoclonal cell lines yielding a recombinant dIFN protein in the amount of >2% of TSP [ 122 ].…”

“…On the other hand, genome-editing technologies make it possible to reduce the screening of genome regions to a search for regions with a high yield of a recombinant protein using reporter genes. The genome regions characterized in terms of accumulation of a recombinant reporter protein (GFP) and the degree of disturbance of other genes (thereby causing a deterioration of, e.g., growth characteristics of cell culture) can serve as the targets for delivering a gene of interest with the help of the knock-in version of genome editing [ 121 ].…”

Increasing the Efficiency of the Accumulation of Recombinant Proteins in Plant Cells: The Role of Transport Signal Peptides

“…Unfortunately, plant expression systems are not free of certain flaws either; the main one is a low yield of recombinant proteins despite all attempts to increase it [ 9 , 10 ]. Several reviews describe the different methods used for enhancing the biosynthesis of transgene-encoded proteins [ 10 , 11 , 12 ].…”