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

Mustapha Aouida1, Dina Aljogol2, Reem Ali1

  • 1College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha 34110, Qatar.

STAR Protocols
|March 14, 2022
PubMed
Summary

This study presents a CRISPR-Cas9 protocol for creating human PRDX1 gene knockout cells. The method efficiently generates knockout cells without antibiotic selection or cell sorting.

Keywords:
Biotechnology and bioengineeringCRISPRCell BiologyCell cultureCell isolationMolecular Biology

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Cell Biology

Background:

  • The PRDX1 gene plays a crucial role in cellular defense mechanisms.
  • Efficient generation of knockout cell lines is essential for functional studies.
  • CRISPR-Cas9 technology offers precise genome editing capabilities.

Purpose of the Study:

  • To establish a robust protocol for generating human PRDX1 knockout cells.
  • To detail a CRISPR-Cas9 based strategy for efficient gene editing.
  • To provide a method that bypasses traditional selection and sorting steps.

Main Methods:

  • Single-guide RNA design, cloning, and transfection into human cells.
  • CRISPR-Cas9 mediated gene editing.
  • T7EI assay for evaluating gene editing efficiency.
  • Single-cell isolation and Sanger sequencing for knockout verification.

Main Results:

  • Successful generation of human PRDX1 knockout cells.
  • Demonstration of efficient DNA editing using the T7EI assay.
  • Verification of indels in one or both alleles by Sanger sequencing.
  • Protocol avoids antibiotic selection and cell sorting.

Conclusions:

  • The described protocol provides an efficient and streamlined method for generating human PRDX1 knockout cells.
  • This approach leverages CRISPR-Cas9 technology for precise gene editing.
  • The protocol is advantageous due to its efficiency and avoidance of antibiotic selection and cell sorting.