Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP-SETDB1 Complex for Enhancing Difficult-to-Express...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Emerging biotherapeutics often present challenges in high-yield production due to difficulties in expression.
• Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical manufacturing, necessitating strategies for improved productivity.

Purpose of the Study:

• To identify novel genetic targets for enhancing the production of difficult-to-express (DTE) proteins, specifically bispecific antibodies (bsAbs).
• To investigate the role of histone methylation in regulating transgene expression and protein productivity in CHO cells.

Main Methods:

• Genome-wide CRISPR knockout screening using fluorescence-activated cell sorting (FACS) in bsAb-producing CHO cells.
• Analysis of gene targets associated with H3K9me3-mediated transcriptional repression, specifically ATF7IP and SETDB1.
• Evaluation of cell growth, protein productivity, H3K9me3 levels, and transgene expression following gene perturbation.

Main Results:

• The screen identified ATF7IP and SETDB1 as key regulators of DTE protein production.
• Knockout of the ATF7IP-SETDB1 complex in bsAb-producing CHO cells suppressed cell growth but increased productivity by up to 2.7-fold.
• Perturbation of ATF7IP-SETDB1 decreased H3K9me3 levels, increased transgene expression, and enhanced monoclonal antibody (mAb) production by up to 3.9-fold without impacting product quality.

Conclusions:

• The ATF7IP-SETDB1 complex is a promising target for enhancing biotherapeutic production in CHO cells.
• Targeting histone methylation pathways, specifically H3K9me3, can unlock transgene expression and significantly improve yields of bsAbs and mAbs.
• Genome-wide CRISPR screening is an effective approach for identifying novel targets to optimize bioprocesses for DTE proteins.