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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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
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author Flumens, Donovan
Campillo-Davo, Diana
Janssens, Ibo
Roex, Gils
De Waele, Jorrit
Anguille, Sébastien
Lion, Eva
author_facet Flumens, Donovan
Campillo-Davo, Diana
Janssens, Ibo
Roex, Gils
De Waele, Jorrit
Anguille, Sébastien
Lion, Eva
author_sort Flumens, Donovan
collection PubMed
description 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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spelling pubmed-99438612023-02-23 One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation Flumens, Donovan Campillo-Davo, Diana Janssens, Ibo Roex, Gils De Waele, Jorrit Anguille, Sébastien Lion, Eva STAR Protoc Protocol 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. Elsevier 2023-02-10 /pmc/articles/PMC9943861/ /pubmed/36853667 http://dx.doi.org/10.1016/j.xpro.2023.102112 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Flumens, Donovan
Campillo-Davo, Diana
Janssens, Ibo
Roex, Gils
De Waele, Jorrit
Anguille, Sébastien
Lion, Eva
One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title_full One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title_fullStr One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title_full_unstemmed One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title_short One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
title_sort one-step crispr-cas9-mediated knockout of native tcrαβ genes in human t cells using rna electroporation
topic Protocol
url 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
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