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.
- RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012). , &
- CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010). &
- CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321–327 (2011). &
- One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013). et al.
- Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013). et al.
- Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013). et al.
- RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013). , , , &
- RNA-programmed genome editing in human cells. Elife 2, e00471 (2013). et al.
- Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013). et al.
- Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). et al.
- RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). et al.
- Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013). , , &
- Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics doi:10.1534/genetics.113.152710 (24 May 2013). et al.
- A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). et al.
- FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012). et al.
- Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011). , , &
- Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008). et al.
- An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011). et al.
- Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011). et al.
- Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211–11216 (1995). et al.
- Supplementary Text and Figures (61 MB)
Supplementary Figures 1–14, Supplementary Note and Supplementary Methods
- Supplementary Table 1 (37 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
- Supplementary Table 2 (53 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