High-frequency Off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics*
  • Endonucleases / genetics*
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Molecular Sequence Data
  • Mutagenesis / genetics*

Substances

  • CRISPR-Associated Proteins
  • Endonucleases