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Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells.

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.

SUBMITTER: Matsui S

PROVIDER: S-EPMC11342178 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells.

Matsui Satoshi S Shiley Joseph R JR Granitto Marissa M Ludwig Katie K Buckley Morgan M Koigi Sandra S Mirizio Gerardo G Hu Yueh-Chiang YC Mayhew Christopher N CN Iwafuchi Makiko M

STAR protocols 20240731 3

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 pla ...[more]

PMID: 39083383

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