Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.
Bak
Bax
CHO
CRISPRi
Casp3
apoptosis
caspase
mitochondrial membrane integrity
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
07 2019
07 2019
Historique:
received:
17
10
2018
revised:
04
02
2019
accepted:
14
03
2019
pubmed:
19
3
2019
medline:
3
7
2020
entrez:
19
3
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30883679
doi: 10.1002/bit.26969
pmc: PMC6545153
mid: NIHMS1018956
doi:
Substances chimiques
bcl-2 Homologous Antagonist-Killer Protein
0
bcl-2-Associated X Protein
0
CRISPR-Associated Protein 9
EC 3.1.-
Caspase 3
EC 3.4.22.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1813-1819Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM119850
Pays : United States
Informations de copyright
© 2019 Wiley Periodicals, Inc.
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