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

Details

• Alternative Title:
  Nat Biotechnol
• Personal Author:
  Fu, Yanfang ; Foden, Jennifer A. ; Khayter, Cyd ; Maeder, Morgan L. ; Reyon, Deepak ; Joung, J. Keith ; Sander, Jeffry D.
• 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.
• Subjects:
  Article Base Sequence CRISPR-Associated Proteins Endonucleases Genetic Engineering HEK293 Cells Humans K562 Cells Molecular Sequence Data Mutagenesis
• Source:
  Nat Biotechnol. 31(9):822-826.
• Pubmed ID:
  23792628
• Pubmed Central ID:
  PMC3773023
• Document Type:
  Journal Article
• Funding:
  DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; P50 HG005550/HG/NHGRI NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States
• Volume:
  31
• Issue:
  9
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:8d457669d7ee59ccbcb4beee9b7a5365daa9fc5419e790927012f50bcda7a760
• Download URL:
  https://stacks.cdc.gov/view/cdc/22517/cdc_22517_DS1.pdf
• File Type:
  [PDF - 337.75 KB ]