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

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.

Generation of heritable germline mutations in the jewel waspNasonia vitripennisusing CRISPR/Cas9

“…caltech.edu) to confirm cn RNA expression of the putative target regions 7,10 . Secondly, we searched both sense and antisense strands of the cn exon sequences of interest for the presence of the NGG protospacer-adjacent motifs (PAMs) utilizing CHOPCHOP v2 software 29 and local sgRNA Cas9 package 30 . Thirdly, to minimize potential off-target effects, we confirmed specificity of our sgRNAs using publicly available bioinformatic tools 31 and selected the most specific sgRNAs within our specified target region.…”

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…Moreover, even with the high specificity of the techniques, the nuclease might still cleave additional sites that are similar to the target sites potentially causing unwanted mutations, known as off-target effects, which is one of the issues of SDNs in general and should be carefully monitored Fichtner et al, 2014;Petersen and Niemann, 2015b). Software tools are being developed to minimize the probability of off-target effects (Naito et al, 2014;Xie et al, 2014), however, the production of an organism with the desired mutation might still require a screening process to eliminate undesirable traits. Nonetheless, compared to conventional transgenesis or induced mutagenesis, the SDNs provide a much less laborious and more straightforward approach (Lusser and Davies, 2013;Araki and Ishii, 2015).…”

“…However, with the increased precision of the SDN techniques, unwanted mutations should occur much less often compared to transgenesis or induced mutagenesis Ribarits et al, 2014;Rinaldo and Ayliffe, 2015). The occurrence of off-target effects can further be reduced by improving binding specificity, use of software tools or by in vitro selection of the most appropriate SDN (Hartung and Schiemann, 2014;Naito et al, 2014;Xie et al, 2014). With the advances in whole-genome sequencing, detection of the organisms carrying unwanted genome changes is also becoming more straightforward Araki and Ishii, 2015).…”

<italic>Mup</italic>-knockout mice generated through CRISPR/Cas9-mediated deletion for use in urinary protein analysis

“…Then, the Cas9 and sgRNA1 or sgRNA2 constructs were electroporated into mouse fibroblast NIH-3T3 cells. And 72 h after eletroporation, T7E1 assay [13] was carried out to evaluate the issue of off-target. Among the 10 most likely off-targeting sites for both sgRNAs (Supplementary Table S3), T7E1 assay revealed no off-target digestion by Cas9 (Supplementary Fig.…”