Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana

Abstract

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.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA

“…After 10 biolistic transformations, the largest number of knock-ins in the target site, six lines, were obtained using the pIFN (H3.3).3 genetic construct, while only three were obtained using the pIFN (H3.3).2 construct. The production of lines with knock-in is described in more detail in our previous work [ 19 ].…”

“…Such sites are necessary for excising the knock-in template from the plasmid and its linearization. The presence of this sites significantly increases the frequency of knock-ins [ 15 , 18 , 19 ]. However, in some cases, apparently, the template is not excised from the plasmid or is not completely excised, which leads to the insertion of additional fragments of plasmid DNA into the plant genome.…”

“…To increase the frequency of the integration of target genes to the target region, they were flanked by a fragment recognized by Cas9, which ensured their excision from the plasmid in the cell in the form of a linear structure. A detailed description of the creation of this plasmids is described in our previous works [ 19 , 24 ].…”

“…After a series of biolistic transformations, three lines were obtained using a knock-in template carrying flanking DNA homologous to the insertion site in the plant genome and presumably integrated into the genome by the HDR mechanism. Two lines were obtained using a knock-in template without flanking DNA and presumably integrated into the genome by the NHEJ mechanism [ 19 ].…”

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

“…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.…”