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

Mustapha Aouida; Dina Aljogol; Reem Ali; Dindial Ramotar

doi:10.1016/j.xpro.2022.101216

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

Mustapha Aouida^*, Dina Aljogol, Reem Ali, Dindial Ramotar^*

^*Corresponding author for this work

Biological and Biomedical Sciences

Hamad bin Khalifa University

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

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.

Original language	English
Article number	101216
Journal	STAR Protocols
Volume	3
Issue number	1
DOIs	https://doi.org/10.1016/j.xpro.2022.101216
Publication status	Published - 18 Mar 2022

Keywords

Biotechnology and bioengineering
CRISPR
Cell Biology
Cell culture
Cell isolation
Molecular Biology

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10.1016/j.xpro.2022.101216

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abstract = "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.",

keywords = "Biotechnology and bioengineering, CRISPR, Cell Biology, Cell culture, Cell isolation, Molecular Biology",

author = "Mustapha Aouida and Dina Aljogol and Reem Ali and Dindial Ramotar",

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AU - Aouida, Mustapha

AU - Aljogol, Dina

AU - Ali, Reem

AU - Ramotar, Dindial

PY - 2022/3/18

Y1 - 2022/3/18

N2 - 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.

AB - 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.

KW - Biotechnology and bioengineering

KW - CRISPR

KW - Cell Biology

KW - Cell culture

KW - Cell isolation

KW - Molecular Biology

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JO - STAR Protocols

JF - STAR Protocols

IS - 1

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ER -

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

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Keywords

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