Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production

Abstract

RNA interference (RNAi) is an effective mechanism for inhibiting gene expression at the post-transcriptional level. Expression of a messenger RNA (mRNA) can be inhibited by a ∼22-nucleotide (nt) small interfering (si)RNA with the corresponding reverse complementary sequence. Typically, a duplex of siRNA, composed of the desired siRNA and a passenger strand, is processed from a short hairpin RNA (shRNA) precursor by Dicer. Subsequently, one strand of the siRNA duplex is associated with Argonaute (Ago) protein for RNAi. Although RNAi is widely used, the off-target effect induced by the passenger strand remains a potential problem. Here, based on current understanding of endogenous precursor microRNA (pre-miRNA) hairpins, called Ago-shRNA and m 7 G-capped pre-miRNA, we discuss the principles of shRNA designs that produce a single siRNA from one strand of the hairpin.

Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

“…However, only ~25% of human miRNA are chosen accordingly to both conditions and as much as ~25% totally eludes these rules (Medley, Panzade, & Zinovyeva, 2020). Likewise, the strand of shRNA-derived siRNA designed to be a passenger strand can also be loaded into Ago (Sheng et al, 2020), thus doubling the number of potential off-targets. Although sh- and siRNAs are designed to have an array of 19-20 nucleotides complementary to sequences within specific targets, the complementarity of a seed region comprising nucleotides 2-7/8 of a guide strand and a hexamer or heptamer in a 3’UTR of mRNA may be sufficient for off-target activity (Birmingham et al, 2006).…”

“…The example of SHC016, which targets SNRPD3, a gene essential for cell survival, highlights that control shRNAs may elicit strong off-target effects that invalidate reliable analysis of RNAi data. Despite continued efforts to improve shRNA scaffolds (Sheng et al, 2020), the chances for elimination or significant reduction of RNAi off-target activity while preserving on-target efficiency are low. Processing of shRNA and its interactions with binding partners are influenced by many poorly understood variables, but most importantly siRNA mimics a natural miRNA mechanism, which by definition does not show high specificity – one miRNA usually silences from several to several dozen transcripts (Liu & Wang, 2019).…”

“…However, inefficient transfection of many cell lines as well as transience of siRNA activity led to the development of short hairpin RNA (shRNA) expression vectors including retro- and lentiviral vectors (Paddison, Caudy, Sachidanandam, & Hannon, 2004). The expression cassette coding for a given shRNA is stably integrated into DNA of transduced cells and the transcribed shRNA, which mimics pre-miRNA, is processed by Dicer to an siRNA duplex (Sheng, Flood, & Xie, 2020).…”

Artificial miRNAs as therapeutic tools: Challenges and opportunities

“…Vector‐based RNAi triggers vary in many ways. RNA polymerase III (Pol III)‐driven shRNAs resemble endogenous miRNA precursors (pre‐miRNAs), which have a characteristic stem‐loop structure (Ma et al, 2014; Sheng et al, 2020). A target‐specific siRNA sequence forms the stem of an ~60 nucleotide (nt) shRNA and is released as a result of single‐step processing in the cytoplasm by the RNase DICER.…”

Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

“…3 An expression cassette coding for a given shRNA is stably integrated into the DNA of transduced cells, and the transcribed shRNA, which mimics pre-miRNA, is processed by Dicer to an siRNA duplex. 4 The need for unified research tools has triggered the development of shRNA libraries of sequences that silence individual genes in the same shRNA backbone. Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets.…”