CRISPR-Cas9 editing of synaptic genes in human embryonic stem cells for functional analysis in induced human neurons.

Autor:	Houcek A; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA., Ma ZZ; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA. Electronic address: zhenzhong.ma@vanderbilt.edu., Trauterman B; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA., Uzay B; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA., Monteggia LM; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA., Kavalali ET; Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA. Electronic address: ege.kavalali@vanderbilt.edu.
Jazyk:	angličtina
Zdroj:	STAR protocols [STAR Protoc] 2024 Jun 21; Vol. 5 (2), pp. 103089. Date of Electronic Publication: 2024 May 24.
DOI:	10.1016/j.xpro.2024.103089
Abstrakt:	Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons. Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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