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@INPROCEEDINGS{Pouramini:917380,
      author       = {Pouramini, P and Koller, Robert and Huber, Gregor and
                      Fischbach, Andreas and Birnisdóttir, Björg and Björnsson,
                      JM and Hensel, G},
      title        = {{T}argeted knockout of barley endosperm-specific storage
                      proteins as a prerequisite for molecular farming purposes},
      reportid     = {FZJ-2023-00594},
      year         = {2022},
      abstract     = {The concept for producing valuable proteins in plants is
                      known as molecular farming. Besides transient expression
                      using viral vector systems in tobacco leaves, the cereal
                      grain is a natural bioreactor ideal for storing proteins
                      under ambient conditions. Therefore, the cereal grain
                      provides a cost-effective, easily scalable expression system
                      for producing high-value proteins in the starchy endosperm.
                      CRISPR-mediated targeted knockout of barley Hordein B1
                      family members was achieved, and Sanger and deep amplicon
                      sequencing genotyped mutant plants. ELISA and Western
                      blotting performed the abundance and quantification of the
                      recombinant protein overexpressed in wildtype and mutant
                      background. An automated phenotyping device evaluated the
                      morphological changes of the mutant grains. To increase
                      protein yield and overcome the competition between
                      endogenous storage protein accumulation and the high-value
                      protein, a targeted knockout of the Hordein B1 family
                      members by using Cas9 technology was performed. The
                      recombinant human Epidermal Groth Factor (EGF) was
                      overexpressed in wildtype and horb1 mutant background as a
                      proof-of-concept. Grains of the resulting horb1 mutants
                      showed altered biometric traits, reduced total protein and
                      hordein content, and delayed germination behaviour compared
                      to wild type segregants. Western blotting and ELISA results
                      confirmed a high EGF accumulation in the horb1 mutant
                      background. In conclusion, the strategy of higher abundance
                      of a recombinant protein in barley grains was successfully
                      demonstrated by reducing endogenous storage protein
                      accumulation. Further fine-tuning is required to minimize
                      the germination disadvantage.},
      month         = {Aug},
      date          = {2023-08-28},
      organization  = {Botanik-Tagung International
                       Conference of the German Society for
                       Plant Sciences 2022, Bonn (Germany), 28
                       Aug 2023 - 1 Sep 2023},
      subtyp        = {After Call},
      cin          = {IBG-2},
      cid          = {I:(DE-Juel1)IBG-2-20101118},
      pnm          = {2171 - Biological and environmental resources for
                      sustainable use (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2171},
      typ          = {PUB:(DE-HGF)6},
      url          = {https://juser.fz-juelich.de/record/917380},
}