The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to 
genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. 
Here, we characterize SpCas9 targeting specificity in human cells to inform the 
selection of target sites and avoid off-target effects. Our study evaluates >700 
guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted 
genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates 
mismatches between guide RNA and target DNA at different positions in a 
sequence-dependent manner, sensitive to the number, position and distribution of 
mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA 
methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize 
off-target modification. To facilitate mammalian genome engineering 
applications, we provide a web-based software tool to guide the selection and 
validation of target sequences as well as off-target analyses.