The CRISPR/Cas or Cas9/guide RNA system is a newly developed, easily engineered and highly effective tool for gene targeting; it has considerable off-target effects in cultured human cells and in several organisms. However, the Cas9/guide RNA target site is too short for existing alignment tools to exhaustively and effectively identify potential off-target sites. CasOT is a local tool designed to find potential off-target sites in any given genome or user-provided sequence, with user-specified types of protospacer adjacent motif, and number of mismatches allowed in the seed and non-seed regions. http://eendb.zfgenetics.org/casot/ CONTACT: zfgenetics@gmail.com or bzhang@pku.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.