Knockout of Sf-Caspase-1 generates apoptosis-resistant Sf9 cell lines: Implications for baculovirus expression.
CRISPR-Cas9
Sf-Caspase-1
Sf9
apoptosis
baculovirus
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Jul 2022
Jul 2022
Historique:
revised:
22
03
2022
received:
29
09
2021
accepted:
01
04
2022
pubmed:
7
4
2022
medline:
16
7
2022
entrez:
6
4
2022
Statut:
ppublish
Résumé
The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing β-galactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.
Identifiants
pubmed: 35384325
doi: 10.1002/biot.202100532
doi:
Substances chimiques
Caspases
EC 3.4.22.-
Caspases, Effector
EC 3.4.22.-
Caspase 1
EC 3.4.22.36
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2100532Informations de copyright
© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
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