sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites

PLoS One. 2014 Jun 23;9(6):e100448. doi: 10.1371/journal.pone.0100448. eCollection 2014.


Although the CRISPR/Cas9/sgRNA system efficiently cleaves intracellular DNA at desired target sites, major concerns remain on potential "off-target" cleavage that may occur throughout the whole genome. In order to improve CRISPR-Cas9 specificity for targeted genome editing and transcriptional control, we describe a bioinformatics tool "sgRNAcas9", which is a software package developed for fast design of CRISPR sgRNA with minimized off-target effects. This package consists of programs to perform a search for CRISPR target sites (protospacers) with user-defined parameters, predict genome-wide Cas9 potential off-target cleavage sites (POT), classify the POT into three categories, batch-design oligonucleotides for constructing 20-nt (nucleotides) or truncated sgRNA expression vectors, extract desired length nucleotide sequences flanking the on- or off-target cleavage sites for designing PCR primer pairs to validate the mutations by T7E1 cleavage assay. Importantly, by identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of sgRNA for genome editing applications. sgRNAcas9 software package is publicly available at BiooTools website ( under the terms of the GNU General Public License.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Base Sequence
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Computational Biology / methods*
  • DNA Cleavage
  • Humans
  • Molecular Sequence Data
  • RNA Editing / genetics*
  • RNA, Guide / genetics*
  • Sequence Homology, Nucleic Acid
  • Software*


  • RNA, Guide

Grant support

The Project was supported by the National Natural Science Foundation of China (Grant No. 31301226) and the Chinese Academy of Sciences (CAS) Knowledge Innovation Program (KSXC2-EW-R-07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.