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Nat Biotechnol. 2013 Sep;31(9):822-6. doi: 10.1038/nbt.2623. Epub 2013 Jun 23.

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

Author information

1
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [4] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

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.

Comment in

PMID:
23792628
PMCID:
PMC3773023
DOI:
10.1038/nbt.2623
[Indexed for MEDLINE]
Free PMC Article

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