SgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.

Shengsong Xie,Bin Shen,Yonglian Zhang,Chaobao Zhang,Xingxu Huang,Arkady B Khodursky

doi:10.1371/journal.pone.0100448

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.

Highlights

Development of tools for targeted genome editing and regulation of gene expression has significantly expanded our ability to elucidate the mechanisms of interesting biological phenomena, and to engineer desirable biological systems
The CRISPR-CRISPR-associated nuclease 9 (Cas9) system has been successfully used in gene targeting of different species, including the monkey, human induced pluripotent stem cells, the mouse, the rat, the zebrafish and the fly [9,10,11,12,13,14,15,16,17,18,19,20,21,22]
We describe sgRNAcas9: a software package that can be applied to search rapidly for CRISPR target sites, and analyze the PLOS ONE | www.plosone.org sgRNAcas9:A Tool for Fast Designing CRISPR single guide RNA (sgRNA) with High Specificity potential off-target cleavage sites of CRISPR-Cas9 simultaneously

Summary

Introduction

Development of tools for targeted genome editing and regulation of gene expression has significantly expanded our ability to elucidate the mechanisms of interesting biological phenomena, and to engineer desirable biological systems. Online tools only evaluate sgRNA potential off-target cleavage sites for a given species’ genome. Candidate CRISPR target sequences with high specificity will be provided to design a sgRNA expression vector.

Results

Conclusion