Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells

CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for...

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Autores principales: Geyer, Fabian, Geyer, Maximilian, Klapproth, Sarah, Wolff, Klaus-Dietrich, Nieberler, Markus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10339245/
https://www.ncbi.nlm.nih.gov/pubmed/37421616
http://dx.doi.org/10.1016/j.xpro.2023.102366
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author Geyer, Fabian
Geyer, Maximilian
Klapproth, Sarah
Wolff, Klaus-Dietrich
Nieberler, Markus
author_facet Geyer, Fabian
Geyer, Maximilian
Klapproth, Sarah
Wolff, Klaus-Dietrich
Nieberler, Markus
author_sort Geyer, Fabian
collection PubMed
description CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.
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spelling pubmed-103392452023-07-14 Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells Geyer, Fabian Geyer, Maximilian Klapproth, Sarah Wolff, Klaus-Dietrich Nieberler, Markus STAR Protoc Protocol CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines. Elsevier 2023-07-07 /pmc/articles/PMC10339245/ /pubmed/37421616 http://dx.doi.org/10.1016/j.xpro.2023.102366 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Geyer, Fabian
Geyer, Maximilian
Klapproth, Sarah
Wolff, Klaus-Dietrich
Nieberler, Markus
Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title_full Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title_fullStr Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title_full_unstemmed Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title_short Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells
title_sort protocol for generating monoclonal crispr-cas9-mediated knockout cell lines using rnps and lipofection in hnscc cells
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10339245/
https://www.ncbi.nlm.nih.gov/pubmed/37421616
http://dx.doi.org/10.1016/j.xpro.2023.102366
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