Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells
Year: 2024
Type: article
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)
Source: STAR Protocols
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
Funders: Cincinnati Children's Hospital Medical Center, Uehara Memorial Foundation, Cincinnati Children’s Research Foundation, Japan Society for the Promotion of Science London, National Institutes of Health, Japan Society for the Promotion of Science, National Institute of General Medical Sciences, National Institute of Diabetes and Digestive and Kidney Diseases
OpenAlex ID: W4401190302