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

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.