One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation

To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8(+) T cells followed by electropor...

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Detalles Bibliográficos
Autores principales: Flumens, Donovan, Campillo-Davo, Diana, Janssens, Ibo, Roex, Gils, De Waele, Jorrit, Anguille, Sébastien, Lion, Eva
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9943861/
https://www.ncbi.nlm.nih.gov/pubmed/36853667
http://dx.doi.org/10.1016/j.xpro.2023.102112
Descripción
Sumario:To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8(+) T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRβ constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.

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