Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells.
STAR Protoc
; 5(3): 103221, 2024 Sep 20.
Article
em En
| MEDLINE
| ID: mdl-39083383
ABSTRACT
Inducible loss-of-function strategies are crucial for understanding gene function. However, creating inducible, multiple-gene knockout models is challenging and time-consuming. Here, we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs, designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid, and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Inativação Gênica
/
Células-Tronco Pluripotentes Induzidas
/
Sistemas CRISPR-Cas
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RNA Guia de Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
STAR Protoc
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos