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Updated: Aug 11, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity
Daniel M Sapozhnikov1, Moshe Szyf2
1Department of Pharmacology and Therapeutics, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada. daniel.sapozhnikov@mail.mcgill.ca.
Abstract:
Off-target mutagenesis of CRISPR/Cas systems must be solved to facilitate safe gene therapy. Here, we report a novel approach, termed "PROTECTOR", to shield known off-target sites by directing the binding of an orthologous nuclease-dead Cas protein to the off-target site to sterically interfere with Cas activity. We show that this method reduces off-target mutation rates of two well-studied guide RNAs without compromising on-target activity and that it can be used in combination with high-fidelity Cas enzymes to further reduce off-target editing. This expands the suite of off-target mitigation strategies and offers an ability to protect off-target sites even when their sequences are fully identical to target sites.
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