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.

Abstract

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 (www.biootools.com) 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*

Substances

  • 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.