Off-targeting remains a central challenge in the application of RNAi knockdown experiments utilizing si- or shRNAs. Especially if it comes to screening in medium- to large scales off-targeting increases cost and efforts by reducing validation rates. Off-targeting means that the used RNAi mediator silences other transcripts in addition to the intended one in a sequence dependent manner. Consequently, observed phenotypes can be due to the knockdown of the intended target, the unintended silenced transcripts or to a combination of both.
Some improvements to increase the specificity of siRNAs have been achieved, e.g. by avoiding the 3’-untranslated regions or seed-region homologies. But a design algorithm that can faithfully predict and prevent off-target effects is presently not available.
In contrast, esiRNA are inherently target-specific due to the complex mixture of many different siRNAs. Pooling of many siRNAs that all share their on-target gene is known to increase specificity because all siRNAs in the pool differ by their sequence-dependent off-target signatures. As a consequence, all individual siRNAs add to the overall silencing of the target, while off-target effects are diluted out (figure 2a). A quantitative correlation between pool complexity and specificity can be seen as well. Measuring the number of off-target transcripts by microarray after transfection of increasingly complex pools of siRNAs conclusively shows this quantitative relationship (figure 2b and 2c).