Off-target mutagenesis is an emerging problem when inducing site-specific DNA breaks for genome editing. Cho et al. used high-throughput DNA sequencing to characterize off-target mutagenesis in human cells during editing by the RNA-guided CRISPR–Cas system. They found that off-target mutagenesis occurs frequently at sites that differ by one nucleotide from the intended target site but substantially less frequently at sites with more than one mismatch. Off-target mutagenesis could be reduced through careful design of the guide RNA sequence and by using alternative nucleases to induce paired single-strand rather than double-strand DNA breaks.