(175s) CRISPR/Cas9-Mediated Genome Editing to Generate Cell Wall-Deficient Tobacco By-2 Cell Lines for Improved Recombinant Protein Production

Plant cells have emerged as a powerful bioproduction platform for fully functional human proteins in a cost-efficient and scalable manner. However, significant challenges persist in the BY-2 cell culture system, including low protein production and secretion, the presence of large vacuoles, and difficulties in cryopreservation. These challenges are largely attributed to the complex plant cell wall structure, composed of interconnected polysaccharides. This project aimed to address these challenges by employing top-down cellular engineering approaches to create novel cellulose-deficient or "animal cell-like" plant cell lines for enhanced therapeutic protein production. Using CRISPR/Cas9 genome editing technology, major cellulose synthase A (CESA) catalytic subunit genes were targeted for inactivation. The morphology, cell wall structural composition, cell growth, bioproduction properties, and transcriptomics of the knockout BY-2 cells were characterized. The knockout BY-2 cell line with the CESA3 gene inactivated exhibited a significant reduction in cellulose content (40.2% reduction) and a notable change in cell wall monosaccharide composition. Distinct phenotypic differences were observed in the knockout BY-2 calli compared to wild-type BY-2 callus, including a more compact texture and crystalline white morphology. Transcriptome analysis revealed significant impacts on cell wall biosynthetic genes in the knockout BY-2 cells. This study demonstrates the feasibility of plant cell wall engineering to create new plant cell lines potentially better suited for the production of recombinant proteins.