alexa High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Clinical Research

Clinical Research

Journal of Clinical Research & Bioethics

Author(s): Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D,

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Abstract Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
This article was published in Nat Biotechnol and referenced in Journal of Clinical Research & Bioethics

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