sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites

By Shengsong Xie, Bin Shen, Chaobao Zhang, Xingxu Huang, Yonglian Zhang. PLoS ONE, 2014

385 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/71c7acfbf8ad0c58143a11c18ca5f3aa21c4e6d1

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Citations in the corpus

Target specificity of the CRISPR-Cas9 system (2014, neutral): “In addition, some tools provide support beyond finding potential guides, such as sequences of oligonucleotides for sgRNA construction [78–80] or primers for validation of cleavage at the target site [75,78].”
Biology and Diseases of Mice (2015, neutral): “websites are available to assist in identifying appropriate DNA sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (Xie et al., 2014; Sander et al., 2010; Bae et al., 2014; Reyon et al., 2011; Herscovitch et al., 2012; Wolfson, 2013”
Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR (2016, background): “Although published tools and scoring systems allow ranking sequences by specificity [10–22] and efficiency [2, 8, 23–25], they are usually limited to a handful of genomes and only few evidence-based recommendations exist to optimize offtarget search parameters and on-target efficiency.”
Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites (2015, neutral): “…(Aach et al., 2014; Cradick et al., 2014; Doench et al., 2014; Bae et al., 2014a; Heigwer et al., 2014; Hsu et al., 2013; Lei et al., 2014; Montague et al., 2014; O'Brien and Bailey, 2014; Naito et al., 2014; Sander et al., 2010; Upadhyay and Sharma, 2014; Xie et al., 2014; Zhu et al., 2014).”