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Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference
Author(s) -
Xiong Kai,
Marquart Kim Fabiano,
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
Publication year - 2019
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26969
Subject(s) - chinese hamster ovary cell , microbiology and biotechnology , repressor , biology , crispr , psychological repression , apoptosis , cas9 , rna interference , downregulation and upregulation , cell culture , chemistry , gene , gene expression , genetics , rna
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.