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Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference |
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| Authors: | Kai Xiong,Kim Fabiano Marquart,Karen Julie la Cour Karottki,Shangzhong Li,Isaac Shamie,Jae Seong Lee,Signe Gerling,Nan Cher Yeo,Alejandro Chavez,Gyun Min Lee,Nathan E. Lewis,Helene Faustrup Kildegaard |
| Affiliation: | 1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark Xiong, 2. Marquart, and 3. Karottki have contributed equally to this work.;4. Department of Pediatrics, University of California, San Diego, California Department of Bioengineering, University of California, San Diego, California The Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, California;5. Department of Pediatrics, University of California, San Diego, California The Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, California;6. Department of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea;7. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark;8. Department of Pharmacology and Toxicology, Precision Medicine Institute, University of Alabama, Birmingham, Alabama;9. Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York;10. Department of Pediatrics, University of California, San Diego, California |
| Abstract: | Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB–MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system. |
| Keywords: | apoptosis Bak Bax Casp3 caspase CHO CRISPRi mitochondrial membrane integrity |