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Related Experiment Video

Updated: Apr 22, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Improved genome editing in human cell lines using the CRISPR method.

Ivan M Munoz¹, Piotr Szyniarowski¹, Rachel Toth¹

¹Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.

|October 11, 2014

Summary

This summary is machine-generated.

Researchers refined the Cas9/CRISPR genome editing system for improved gene disruption and analysis. New methods allow Cas9 elimination and gene re-introduction, enabling off-target effect assessment and structure-function studies.

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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

The Cas9/CRISPR system is a widely used genome editing tool.
It functions by using a single guide RNA (sgRNA) to direct the Cas9 nuclease to a specific DNA sequence, inducing a double-strand break that leads to gene disruption through error-prone repair mechanisms.

Purpose of the Study:

To refine the Cas9/CRISPR system for enhanced genome editing capabilities.
To develop methods for assessing off-target effects and performing structure-function analyses.
To establish a controllable gene editing system allowing Cas9 removal and gene re-introduction.

Main Methods:

Development of stable cell lines expressing Cas9.
A PCR-based protocol for sgRNA generation.
A methodology for Cas9 elimination post-disruption and re-introduction of the disrupted gene.
Utilized the Fan1 DNA repair gene as a control for experiments.

Main Results:

Cas9/CRISPR-mediated disruption of the Fan1 gene occurred at approximately 29% frequency.
Disruption resulted in the expected hypersensitivity to genotoxins.
Re-introduction of the Fan1 gene successfully rescued the observed genotoxin hypersensitivity.

Conclusions:

The refined Cas9/CRISPR system offers improved control and analytical capabilities for genome editing.
The developed methodology facilitates the assessment of off-target effects and gene function.
This approach provides a robust platform for studying gene function and validating genome editing outcomes.