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

Y Fu, JA Foden, C Khayter, ML Maeder, D Reyon… - Nature …, 2013 - nature.com
Y Fu, JA Foden, C Khayter, ML Maeder, D Reyon, JK Joung, JD Sander
Nature biotechnology, 2013nature.com
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 …
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.
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