CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.
CRISPR library screening
gene activation
gene knockout
lentiviral vectors
vector titers
Journal
Frontiers in genome editing
ISSN: 2673-3439
Titre abrégé: Front Genome Ed
Pays: Switzerland
ID NLM: 101775540
Informations de publication
Date de publication:
2023
2023
Historique:
received:
07
05
2023
accepted:
03
07
2023
medline:
31
7
2023
pubmed:
31
7
2023
entrez:
31
7
2023
Statut:
epublish
Résumé
Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as
Identifiants
pubmed: 37520398
doi: 10.3389/fgeed.2023.1218328
pii: 1218328
pmc: PMC10373892
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1218328Informations de copyright
Copyright © 2023 Iaffaldano, Marino and Reiser.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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