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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
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Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method
Roberto Ballarino1, Britta A M Bouwman1, Nicola Crosetto2
1Science for Life Laboratory (SciLifeLab), Research Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Methods in Molecular Biology (Clifton, N.J.)
|September 14, 2020
Summary
This study details a method to accurately measure unintended DNA damage from CRISPR gene editing tools. This helps ensure the safety and efficacy of CRISPR therapies by identifying and minimizing off-target effects.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Clustered regularly interspaced palindromic repeat (CRISPR) systems offer powerful therapeutic potential for genetic diseases.
- A significant clinical concern is the induction of unintended off-target (OT) DNA double-strand breaks (DSBs) by CRISPR nucleases.
Purpose of the Study:
- To describe a detailed protocol for quantifying genome-wide OT DSBs induced by CRISPR nucleases.
- To enable the engineering of CRISPR nucleases with minimized OT activity for safer therapeutic applications.
Main Methods:
- The study utilizes the previously developed Breaks Labeling In Situ and Sequencing (BLISS) method.
- CRISPR-BLISS is applied to quantify OT DSBs in cultured cells post-transfection or transduction.
Main Results:
- The CRISPR-BLISS method provides a versatile and scalable approach for assessing CRISPR nuclease activity.
- It allows for the evaluation of multiple guide RNAs across different cell types and time points.
Conclusions:
- CRISPR-BLISS is an effective tool for quantifying genome-wide off-target DNA double-strand breaks.
- This method is crucial for advancing the clinical safety and development of CRISPR-based gene therapies.
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