Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Bioinformatics. 2014 May 15;30(10):1473-5. doi: 10.1093/bioinformatics/btu048. Epub 2014 Jan 24.


Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.

Availability and implementation: Cas-OFFinder free access at

Contact: or

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Base Sequence
  • CRISPR-Associated Protein 9
  • DNA / genetics
  • DNA / metabolism
  • Endonucleases / chemistry*
  • Endonucleases / genetics
  • Genome, Human
  • Genome, Plant
  • Humans
  • Mice
  • RNA, Guide, Kinetoplastida / genetics*
  • RNA, Guide, Kinetoplastida / metabolism
  • Sequence Analysis, DNA / methods*
  • Software*
  • Time Factors


  • Bacterial Proteins
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases