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siRNA off target FYI

Excerpt from:

Trends in Biotechnology
Volume 23, Issue 9 , September 2005, Pages 446-448

The best control for the specificity of RNAi

Mihail Sarov and A. Francis Stewart

Genomics, BioInnovationZentrum, The University of Technology, Dresden, Am Tatzberg 47, 01307 Dresden, Germany

Available online 23 June 2005.

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

The discovery of RNA interference (RNAi) uncovered an unexpected and fundamental aspect of eukaryotic molecular biology, the implications of which are still being determined [1]. Currently, three basic mechanisms are acknowledged: mRNA degradation mediated by small interfering (si)RNA, translational inhibition mediated by microRNA (mi)RNA, and RNAi-provoked chromatin silencing mediated by methylation of nucleosomes. Despite the explosion of research activity in this area, none of these basic mechanisms is well understood. Similarly, the understanding of RNAi mechanisms in cell and developmental biology remains sketchy.

Besides its fundamental appeal, RNAi has attracted interest because of its usefulness as a tool in functional genomics. It can be used to provoke loss of function relatively easily compared with other approaches. The speed, ease and cost-effectiveness of RNAi gene knock-down has led to two main developments. First, large-scale functional screens, previously impractical with other methodologies, are now possible [2]. Second, targeted loss of function, previously limited to the few specialized systems that support efficient homologous recombination, is now widely applicable. These points are particularly relevant to studies with the model organisms Drosophila and Caenorhabditis elegans, as well as the vast untapped potential for reverse genetics with cultured cell lines. Now, both large-scale screens and testing for individual loss of function have entered routine practice.

Specificity issues
The rapidly spreading popularity of RNAi methodology emphasizes the need to understand its strengths and limitations as a tool. The issue of target specificity and off-target effects has been a source of concern since the first applications of RNAi to functional genomics [3]. Notably, work with vertebrate systems unveiled overlap with a general phenomena provoked by double-stranded (ds)RNA, mediated by protein kinase R (PKR), translational inhibition and the interferon response 4 and 5. These widespread, deleterious responses are not related to the nucleotide sequence of the dsRNA and can significantly complicate RNAi loss-of-function studies. The recognition that RNAi is mediated by dsRNAs of less than 30 bps was key to defining the way to use RNAi for vertebrate analysis [6]. Hence, siRNAs (i.e. less than 30 bps) are now used in vertebrate systems. However, the dsRNA response remains a potential hazard for applications of RNAi. This is not the only reason for concluding that RNAi loss-of-function experiments need good controls.

To date, applications of RNAi aim to provoke loss of function by eliciting specific mRNA degradation. Often, mRNA levels are significantly depleted but not abolished. Because production of the target mRNA continues regardless of degradation, and a certain level of translation is expected, RNAi loss-of-function applications are termed ‘knock-down’ to distinguish them from ‘knockouts’ achievable by mutagenesis at the DNA level. An RNAi knock-down is an ongoing balance between the rate of production of the target mRNA and the efficiency of the RNAi-directed mechanism for target mRNA degradation. Because it is a balance, RNAi knock-down experiments are inherently variable. Therefore, they need exacting controls.

Furthermore, RNAi is not limited to targeted mRNA degradation. Two other short dsRNA mechanisms overlap with siRNA-directed mRNA degradation. Of these, inhibition of translation by miRNAs has been the most worrisome for RNAi applications because miRNAs are not perfectly complementary to their targets. Consequently, it has proven difficult to determine the characteristics that define an miRNA, leading to the fear that siRNA designed for one target mRNA might inadvertently convey an miRNA effect on a different target. So little is known about the other known short dsRNA mechanism, which is involved in chromatin silencing, that no predictions as to specificities can safely be made. Because we do not yet understand RNAi well, we cannot depend upon its performance as a tool. Herein lie further reasons for exacting controls....