Details:

Alternative Title:
Nat Biotechnol
Personal Author:
Fu, Yanfang ; Foden, Jennifer A. ; Khayter, Cyd ; Maeder, Morgan L. ; Reyon, Deepak ; ... More +
Fu, Yanfang ; Foden, Jennifer A. ; Khayter, Cyd ; Maeder, Morgan L. ; Reyon, Deepak ; Joung, J. Keith ; Sander, Jeffry D. ; Less -
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.
Subject:
[+]
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 ; P50 HG005550/HG/NHGRI NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; ... More +
DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; P50 HG005550/HG/NHGRI NIH HHS/United States ; P50 HG005550/HG/NHGRI NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:8d457669d7ee59ccbcb4beee9b7a5365daa9fc5419e790927012f50bcda7a760
File Type:

Supporting Files

• 	NIHMS488397-supplement-1.pdf	pdf
  	NIHMS488397-supplement-table_1.pdf	pdf
  	NIHMS488397-supplement-table_2.pdf	pdf
  	nihms488397.nxml	bin
  	nihms488397f1.gif	gif
  	nihms488397f1.jpg	jpeg

More +