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Pouneh Pouramini1, Hildur Björg Birnisdóttir2, Jón Már Björnsson2 and Goetz Hensel3 (1Leibniz Institute of Plant Genetics and Crop Plant Research, Seeland, Germany 2ORF Genetics, Reykjavik, Iceland 3Centre for Plant Genome Engineering, Heinrich-Heine-University, Dusseldorf, Germany)
The concept of 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. 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 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. ELISA and Western blotting confirmed the abundance and quantification of the recombinant protein overexpressed in wild-type and mutant background. Sanger and deep amplicon sequencing confirmed horb1 mutant plants. Grains of the resulting horb1 mutants showed altered biometric traits, reduced total protein and hordein content, and delayed germination behavior compared to wild-type segregants.