Development of a graphical user interface for sgRNAcas9 and its application.

The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).