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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
PubMed

Insights

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.
Keywords:
BLISSCRISPRDNA double-strand breaksOff-targets

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