The PROTECTOR strategy employs dCas orthologs to sterically shield off-target sites from CRISPR/Cas activity

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.

Automated summary

PROTECTOR is a novel approach that uses a nuclease-dead Cas protein to bind to off-target sites and interfere with Cas activity, reducing off-target mutation rates without compromising on-target activity. It can be used in combination with high-fidelity Cas enzymes to further decrease off-target editing, offering an ability to protect off-target sites with identical sequences to target sites.