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

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.

SUBMITTER: Sapozhnikov DM

PROVIDER: S-EPMC9911626 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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

Sapozhnikov Daniel M DM Szyf Moshe M

Scientific reports 20230209 1

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-fideli ...[more]

PMID: 36759683

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