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Related Experiment Video

Updated: Nov 26, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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An efficient and precise method for generating knockout cell lines based on CRISPR-Cas9 system.

Xibin Lu1, Yuhan Guo2, Shu Gu3

  • 1Core Research Facilities Southern University of Science and Technology Shenzhen P. R. China.

Engineering in Life Sciences
|December 11, 2020
PubMed
Summary
This summary is machine-generated.

This study presents an efficient CRISPR-Cas9 gene editing method for creating knockout cell lines. The optimized knock-in strategy significantly reduces the time and effort required for generating genetically modified cells.

Keywords:
CRISPR‐Cas9homologous recombinationknockoutknock‐intargeting efficiency

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 offers improved genome editing over older methods.
  • Screening knockout/knock-in cell clones remains time-consuming and labor-intensive.
  • Guide RNA (gRNA) efficacy and target site variability impact screening efficiency.

Purpose of the Study:

  • To adapt and characterize a targeted knock-in strategy using CRISPR-Cas9.
  • To improve the efficiency of generating single and double knockout cell lines.
  • To establish a faster and more reliable method for obtaining genetically modified cell clones.

Main Methods:

  • Co-transfection of a homology-arm based donor cassette (fluorescence protein and antibiotic resistance gene) with a gRNA expressing unit.
  • Utilized Fluorescence-Activated Cell Sorting (FACS) and antibiotic selection for positive clone enrichment.
  • Confirmed successful targeting through genotyping and protein expression analysis.

Main Results:

  • Achieved over 70% successful targeting efficiency in both single and double knockout experiments.
  • The entire procedure, from transfection to obtaining knockout cell lines, takes less than three weeks.
  • High efficiency was observed in generating targeted cell clones using FACS sorting and antibiotic selection.

Conclusions:

  • The developed CRISPR-Cas9 targeted knock-in strategy significantly enhances the efficiency of generating knockout cell lines.
  • This methodology offers a versatile and rapid approach for creating genetically modified cell clones.
  • The protocol is expected to be broadly applicable across various cell lines for efficient gene editing.