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High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
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Jun 23 2013
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Source: Nat Biotechnol. 31(9):822-826.
[PDF-337.75 KB]
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Alternative Title:Nat Biotechnol
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Description: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.
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Pubmed ID:23792628
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Pubmed Central ID:PMC3773023
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Volume:31
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Issue:9
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