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

Yanfang Fu, Jennifer A Foden, Cyd Khayter, Morgan L Maeder, Deepak Reyon, J Keith Joung, Jeffry D Sander
Source: Nat Biotechnol
Publication Date: (2013)
Issue: 31(9): 822-6
Research Area:
Cancer Research/Cell Biology
Basic Research
Molecular Biology
Cells used in publication:
Species: human
Tissue Origin: blood
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector™ X-Unit
U2OS.EGFP cells... For transfections, 200,000 cells were Nucleofected with the indicated amounts of sgRNA expression plasmid and pJDS246 together with 30 ng of a Td-tomato-encoding plasmid using the SE Cell Line 4D-Nucleofector™ X Kit (Lonza) according to the manufacturer’s protocol. Cells were analyzed 2 days post-transfection using flow cytometry. Transfections for optimizing gRNA/Cas9 plasmid concentration were performed in triplicate and all other transfections were performed in duplicate. For U2OS.EGFP and K562 cells, 2 × 105 cells were transfected with 250 ng of sgRNA expression plasmid or an empty U6 promoter plasmid (for negative controls), 750 ng of Cas9 expression plasmid, and 30 ng of td-Tomato expression plasmid using the 4D Nucleofector System according to the manufacturer’s instructions (Lonza).
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.