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.
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This work was supported by a US National Institutes of Health (NIH) Director's Pioneer Award DP1 GM105378, NIH R01 GM088040, NIH P50 HG005550, Defense Advanced Research Projects Agency (DARPA) W911NF-11-2-0056, and the Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. We thank S.Q. Tsai for helpful discussions and encouragement.
J.K.J. has a financial interest in Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.
Supplementary Figures 1–14, Supplementary Note and Supplementary Methods (PDF 312 kb)
Sequences of oligonucleotides used to generate expression plasmids encoding sgRNAs/variant sgRNAs targeted to sites in the EGFP reporter gene and sgRNAs targeted to six endogenous human gene targets (XLSX 34 kb)
Sequences and characteristics of genomic on- and off-target sites for six RGENs targeted to endogenous human genes and primers and PCR conditions used to amplify these sites (XLSX 48 kb)
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Fu, Y., Foden, J., Khayter, C. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31, 822–826 (2013). https://doi.org/10.1038/nbt.2623
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