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Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interferenceoa mark
  • Xiong, Kai ;
  • Marquart, Kim Fabiano ;
  • la Cour Karottki, Karen Julie ;
  • Li, Shangzhong ;
  • Shamie, Isaac ;
  • Lee, Jae Seong ;
  • Gerling, Signe ;
  • Yeo, Nan Cher ;
  • Chavez, Alejandro ;
  • Lee, Gyun Min ;
  • Lewis, Nathan E. ;
  • Kildegaard, Helene Faustrup
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dc.contributor.authorXiong, Kai-
dc.contributor.authorMarquart, Kim Fabiano-
dc.contributor.authorla Cour Karottki, Karen Julie-
dc.contributor.authorLi, Shangzhong-
dc.contributor.authorShamie, Isaac-
dc.contributor.authorLee, Jae Seong-
dc.contributor.authorGerling, Signe-
dc.contributor.authorYeo, Nan Cher-
dc.contributor.authorChavez, Alejandro-
dc.contributor.authorLee, Gyun Min-
dc.contributor.authorLewis, Nathan E.-
dc.contributor.authorKildegaard, Helene Faustrup-
dc.date.issued2019-07-01-
dc.identifier.urihttps://dspace.ajou.ac.kr/dev/handle/2018.oak/30737-
dc.description.abstractChinese 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.-
dc.description.sponsorshipThe authors like to thank Nachon Charanyanonda Petersen and Karen Kathrine Brøndum for assistance with the FACS and Mads Valdemar Anderson for computational support. We acknowledge support from the Novo Nordisk Foundation (NNF10CC1016517 and NNF16OC0021638) and support from NIGMS (R35 GM119850). AC was funded by the Burroughs Wellcome Career Award for Medical Scientist.-
dc.description.sponsorshipNational Institute of General Medical Sciences, Grant/Award Number: R35 GM119850; Novo Nordisk Foundation, Grant/Award Numbers: NNF10CC1016517, NNF16OC0021638; Burroughs ; Wellcome Career Award for Medical Scientist-
dc.language.isoeng-
dc.publisherJohn Wiley and Sons Inc.-
dc.subject.meshBiopharmaceutical proteins-
dc.subject.meshCasp3-
dc.subject.meshCaspases-
dc.subject.meshChinese Hamster ovary cells-
dc.subject.meshCRISPRi-
dc.subject.meshEndogenous expression-
dc.subject.meshMitochondrial membranes-
dc.subject.meshProtein production-
dc.subject.meshAnimals-
dc.subject.meshApoptosis-
dc.subject.meshbcl-2 Homologous Antagonist-Killer Protein-
dc.subject.meshbcl-2-Associated X Protein-
dc.subject.meshCaspase 3-
dc.subject.meshCHO Cells-
dc.subject.meshCricetulus-
dc.subject.meshCRISPR-Associated Protein 9-
dc.subject.meshCRISPR-Cas Systems-
dc.subject.meshGene Targeting-
dc.titleReduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference-
dc.typeArticle-
dc.citation.endPage1819-
dc.citation.startPage1813-
dc.citation.titleBiotechnology and Bioengineering-
dc.citation.volume116-
dc.identifier.bibliographicCitationBiotechnology and Bioengineering, Vol.116, pp.1813-1819-
dc.identifier.doi10.1002/bit.26969-
dc.identifier.pmid30883679-
dc.identifier.scopusid2-s2.0-85066475656-
dc.identifier.urlhttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-0290-
dc.subject.keywordapoptosis-
dc.subject.keywordBak-
dc.subject.keywordBax-
dc.subject.keywordCasp3-
dc.subject.keywordcaspase-
dc.subject.keywordCHO-
dc.subject.keywordCRISPRi-
dc.subject.keywordmitochondrial membrane integrity-
dc.description.isoatrue-
dc.subject.subareaBiotechnology-
dc.subject.subareaBioengineering-
dc.subject.subareaApplied Microbiology and Biotechnology-
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