Enhanced membrane protein production in HEK293T cells via <i>ATF4</i> gene knockout: A CRISPR-Cas9 mediated approach

• DOI: 10.17305/bb.2024.11519
• Publication Date: 2025-01-21T14:59:07Z
• Authors: Byung-Jo Choi; Ba Reum Kim; Ho Joong Choi; Ok-Hee Kim; Say-June Kim
• Keywords: HEK 293 cells; Gene knockout; Knockout mouse

HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed enhance membrane in by knocking out the ATF4 gene using CRISPR-Cas9 technology. The was edited infecting with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted all-in-one two-vector systems. Genome sequencing productivity of ATF4-knockout (KO) compared wild-type (WT) next-generation (NGS) isolation kit, respectively. Single-cell analysis confirmed editing patterns, NGS verifying intended deletions. Membrane also assessed indirectly via flow cytometry, analyzing expressing Membrane-GFP. Compared WT cells, ATF4-KO exhibited significant increase production, 52.2 ± 19.0% improvement. Gene efficiency between two delivery systems, system demonstrating higher based on T7 endonuclease I assays. Western blot suppression increased expression proteins, including E-cadherin CD63. Quantitative PAGE revealed 77.2 30.6% purified yields, consistent observed enhancements. Flow cytometry Membrane-GFP further demonstrated 22.9 9.7% productivity. In summary, knockout significantly enhances offering potential improvements biopharmaceutical manufacturing enabling more efficient synthesis.