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