(a-c) The indel mutation frequency from high-throughput DNA sequencing of amplified genomic on-target sites and off-target sites () from human cells treated with fCas9 or Cas9 nickase and (a) two sgRNAs (sgRNAs SA1 and SA2) spaced 15 bp apart targeting site A, human genomic locus chr1:21,655,401-21,655,461; (b) two sgRNAs (sgRNAs SB1 and SB2) spaced 24 bp apart targeting site B, human genomic locus chr2:31,485,447-31,485,516; or (c) two sgRNAs (sgRNAs SC1 and SC2) spaced 23 bp apart targeting the site C, human genomic locus chr3:48,747,484-48,747,552. P values are listed in . Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of 83,000 sequences analyzed per off-target sample (). The mock transfection control represents the limit of detection for each site, determined from cells transfected with a GFP expression plasmid and no sgRNA or nuclease expression constructs.