Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method

Part of the Methods in Molecular Biology book series (MIMB, volume 2162)


Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.

Key words

CRISPR Off-targets DNA double-strand breaks BLISS 



We thank Winston Yan and Feng Zhang from the Broad Institute for support in setting up CRISPR-BLISS.


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© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Science for Life Laboratory (SciLifeLab), Research Division of Genome Biology, Department of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden

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