Endogenous mRNAs partially complementary to the scrambled shRNA guide strand

This entry refers to a heterogeneous group of cellular transcripts that are inadvertently regulated by non-targeting control sequences used in RNA interference (RNAi) experiments. Although scrambled shRNAs are designed to lack perfect complementarity to any known gene, they frequently induce off-target effects by binding to mRNAs through partial sequence complementarity [Jackson, A. L., et al. (2003). Nature Biotechnology, 21(6), 635-637]. This binding primarily occurs via the 6-8 nucleotide seed region of the guide strand, mimicking the natural regulatory mechanism of microRNAs (miRNAs) [Birmingham, A., et al. (2006). Nature Methods, 3(3), 199-204]. Such interactions lead to unintended mRNA degradation or translational inhibition, which can significantly alter the cellular transcriptome and proteome [Sigoillot, F. D., et al. (2012). Nature Methods, 9(4), 363-366]. These off-target effects are a major source of false-positive results in functional genomics screens and can cause unexpected cytotoxicity. Furthermore, high-level expression of exogenous shRNAs can saturate the cellular RNA-induced silencing complex (RISC) machinery, interfering with the processing of endogenous miRNAs [Grimm, D., et al. (2006). Nature, 441(7092), 537-541]. This saturation can lead to severe organ toxicity, as demonstrated in murine liver models. Additionally, certain shRNA sequences may trigger innate immune responses through the activation of Toll-like receptors [Bridge, A. J., et al. (2003). Nature Genetics, 34(3), 263-264]. Consequently, these mRNAs are not therapeutic targets but represent a critical technical challenge and safety concern in the development of RNAi-based therapeutics. Understanding and minimizing these interactions is essential for ensuring the specificity and safety of gene-silencing technologies.

Other names

shRNA off-targetsSeed-match off-target mRNAsNon-targeting control off-targetsOff-target transcripts

RNA-induced silencing complex (RISC)-mediated gene silencing via partial seed-sequence complementarity [Jackson, A. L., et al. (2003). Nature Biotechnology, 21(6), 635-637].