A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system

Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verificatio...

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Autores principales: Aouida, Mustapha, Aljogol, Dina, Ali, Reem, Ramotar, Dindial
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8904610/
https://www.ncbi.nlm.nih.gov/pubmed/35284843
http://dx.doi.org/10.1016/j.xpro.2022.101216
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author Aouida, Mustapha
Aljogol, Dina
Ali, Reem
Ramotar, Dindial
author_facet Aouida, Mustapha
Aljogol, Dina
Ali, Reem
Ramotar, Dindial
author_sort Aouida, Mustapha
collection PubMed
description Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.
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spelling pubmed-89046102022-03-10 A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system Aouida, Mustapha Aljogol, Dina Ali, Reem Ramotar, Dindial STAR Protoc Protocol Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting. Elsevier 2022-03-04 /pmc/articles/PMC8904610/ /pubmed/35284843 http://dx.doi.org/10.1016/j.xpro.2022.101216 Text en © 2022 The Author(s) 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
Aouida, Mustapha
Aljogol, Dina
Ali, Reem
Ramotar, Dindial
A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title_full A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title_fullStr A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title_full_unstemmed A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title_short A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system
title_sort simple protocol to isolate a single human cell prdx1 knockout generated by crispr-cas9 system
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8904610/
https://www.ncbi.nlm.nih.gov/pubmed/35284843
http://dx.doi.org/10.1016/j.xpro.2022.101216
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