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

Fu, Yanfang; Foden, Jennifer A.; Khayter, Cyd; Maeder, Morgan L.; Reyon, Deepak; Joung, J. Keith; Sander, Jeffry D.

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

Published Date:

Jun 23 2013

Source:

Nat Biotechnol. 31(9):822-826.

[PDF-337.75 KB]

Details:

Personal Authors:

Fu, Yanfang ; Foden, Jennifer A. ; Khayter, Cyd ; Maeder, Morgan L. ; Reyon, Deepak ; ... More ▼

Keywords:

[+]

Pubmed ID:

23792628

Pubmed Central ID:

PMC3773023

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.

Document Type:

Journal Article

Collection(s):

CDC Public Access

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
R01 GM088040/GM/NIGMS NIH HHS/United States

Supporting Files:

	NIHMS488397-supplement-1.pdf	application/pdf
	NIHMS488397-supplement-table_1.pdf	application/pdf
	NIHMS488397-supplement-table_2.pdf	application/pdf
	nihms488397.nxml	application/octet-stream
	nihms488397f1.gif	image/gif
	nihms488397f1.jpg	image/jpeg

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