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Related Concept Videos

Genetic Screens02:46

Genetic Screens

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Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing  genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which...
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Related Experiment Video

Updated: Apr 20, 2026

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

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A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing.

Charles C Bell1, Graham W Magor, Kevin R Gillinder

  • 1Mater Research, Faculty of Medicine and Biomedical Science, The University of Queensland, Woolloongabba, Queensland, Australia. charles.bell@mater.uq.edu.au.

BMC Genomics
|November 21, 2014
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Summary

This study introduces a high-throughput CRISPR-Cas9 screening method using next-generation sequencing. The new strategy efficiently identifies gene mutations in up to 96 clones simultaneously, saving time and resources.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR-Cas9 enables efficient eukaryotic genome editing.
  • Current methods for screening CRISPR-Cas9 edited clones are often time-consuming and costly.
  • High-throughput screening is needed to accelerate research utilizing genome editing.

Purpose of the Study:

  • To develop a high-throughput screening strategy for CRISPR-Cas9 edited clones.
  • To enable parallel screening of up to 96 clones simultaneously.
  • To overcome limitations of current laborious and expensive screening methods.

Main Methods:

  • Utilized CRISPR-Cas9 for targeted gene disruption in mouse embryonic stem cells.
  • Developed a high-throughput screening strategy employing next-generation sequencing (NGS).
  • Applied the Ion Torrent PGM platform for simultaneous sequencing of up to 96 clones.

Main Results:

  • Successfully screened 67 CRISPR-Cas9 transfected clones for mutations in the Evx1 gene.
  • Identified homozygous, heterozygous, and mixed clones with high accuracy.
  • Demonstrated the capability to detect clonal heterogeneity.

Conclusions:

  • The developed CRISPR-Cas9 screening strategy is cost-effective and time-efficient.
  • This method is widely applicable for generating mutant cell lines and transgenic organisms.
  • The technique provides crucial information on clonal heterogeneity and is adaptable to various sequencing platforms.