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CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

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Related Experiment Video

Updated: Dec 9, 2025

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Published on: December 12, 2017

Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector.

Xiaoling Wang¹, Youjun Wu², Jiing-Kuan Yee³

¹Guangzhou Medical University, Guangzhou, Guangdong, China.

Methods in Molecular Biology (Clifton, N.J.)

|September 14, 2020

Summary

Gene editing tools like CRISPR can cause unintended DNA changes. This study introduces a new method using lentiviral vectors and deep sequencing to accurately map these off-target effects in vivo.

Keywords:

CRISPR/Cas9 Genome editing IDLV Off-target activity

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Last Updated: Dec 9, 2025

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

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Published on: November 14, 2018

Area of Science:

Molecular Biology
Genetics
Bioengineering

Background:

Gene editing technologies like CRISPR, ZFNs, and TALENs offer significant research and therapeutic potential.
A key challenge is predicting and mitigating off-target mutations at unintended genomic locations.
Current in silico prediction methods for off-target activity often lack in vivo accuracy.

Purpose of the Study:

To develop and validate an unbiased screening protocol for mapping gene editing off-target sites.
To improve the accuracy of off-target effect assessment compared to existing in silico methods.

Main Methods:

Utilized integration-defective lentiviral vectors (IDLV) for unbiased screening.
Employed deep sequencing to identify and map off-target cleavage events.
Developed a novel protocol for in vivo off-target site detection.

Main Results:

Successfully mapped off-target sites generated by gene editing nucleases.
Demonstrated the capability of the IDLV-based deep sequencing approach for comprehensive off-target analysis.
Provided a more accurate assessment of in vivo cleavage activity at unintended genomic loci.

Conclusions:

The presented IDLV and deep sequencing protocol offers a robust and unbiased method for identifying gene editing off-target effects.
This approach enhances the safety assessment of gene editing tools for research and therapeutic applications.
Accurate mapping of off-target sites is crucial for the responsible development of gene editing technologies.