Summary: 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/.Contact: y-naito@dbcls.rois.ac.jpSupplementary information: Supplementary data are available at Bioinformatics online.

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Short interfering RNA (siRNA) may down-regulate many unintended genes whose transcripts possess complementarity to the siRNA seed region, which contains 7 nt. The capability of siRNA to induce this off-target effect was highly correlated with the calculated melting temperature or standard free-energy change for formation of protein-free seed duplex, indicating that thermodynamic stability of seed duplex formed between the seed and target is one of the major factor in determining the degree of off-target effects. Furthermore, unlike intended gene silencing (RNA interference), off-target effect was completely abolished by introduction of a G:U pair into the seed duplex, and this loss in activity was completely recovered by a second mutation regenerating Watson–Crick pairing, indicating that seed duplex Watson–Crick pairing is also essential for off-target gene silencing. The off-target effect was more sensitive to siRNA concentration compared to intended gene silencing, which requires a near perfect sequence match between the siRNA guide strand and target mRNA.