UNLABELLED: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼ 20 nt targeted sequence. The target sequence requirements are twofold. First, the 5'-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. AVAILABILITY: Freely available at http://crispr.dbcls.jp/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA 'specificity' design rules.

BACKGROUND: RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value. RESULTS: We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5 degrees C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects. CONCLUSION: siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5 degrees C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at http://siDirect2.RNAi.jp/.