CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain

Abstract

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

StresSeed: The Unfolded Protein Response During Seed Development

“…Whether the protein folding machinery and UPR sensors can be manipulated with the aim of improving seed protein quality and quantity constitutes an important challenge, also in light of the use of seeds for the production of recombinant pharmaceuticals ( Arcalis et al, 2014 ; Panting et al, 2021 ). As it has been underlined ( Cao et al, 2022 ), the loss of storage protein soon after synthesis opens perspectives for possible crop productivity improvement by conventional breeding or genetic engineering.…”

Leveraging the sugarcane CRISPR/Cas9 technique for genetic improvement of non-cultivated grasses

“…This newly developed variant holds potential to target the genomic DNA without requiring PAM and might be declared as nearly PAM-less variant. Thereafter, Ren et al (2021) have reported that SpRY remained equally effective in rice by successfully targeting a large number of NNN PAM sites (NAN/NGN/NCN/NTN). Contrastingly, it was observed that Cas9 was unable to edit a number of relaxed PAM sites and was pronouncedly less efficient in comparison to SpRY for non-canonical PAM sites.…”

Genome editing to re-domesticate and accelerate use of barley crop wild relatives

“…Use of CRISPR/Cas has also increased knowledge of lignin modifications (Lee et al, 2021), vitamin E biosynthesis (Zeng et al, 2020) and protein functions (Galli et al, 2022). Noteworthy is an approach by Panting et al (2021) in which barley was modified for recombinant protein production. To RD barley from its progenitor, we conclude that four major traits would lead to a good foundation.…”