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

Abstract

In parallel with the expansion of RNA interference techniques evidence has been accumulating that RNAi analyses may be seriously biased due to off-target effects of gene-specific shRNAs. Our work points to another possible source of misinterpretations of shRNA-based data – off-target effects of non-targeting shRNA. We found that one such control for the MISSION® library (commercialized TRC library), SHC016, is cytotoxic. Using a lentiviral vector with inducible expression of SHC016 we proved that this shRNA induces apoptosis in murine cells and, depending on p53 status, senescence or mitotic catastrophe in human tumor cell lines. We identified SNRPD3, a core spliceosomal protein, as a major SHC016 target in several cell lines and confirmed in A549 and U251 cell lines that CRISPRi-knockdown of SNRPD3 mimics the effects of SHC016 expression. Our finding disqualifies non-targeting SHC016 shRNA and adds a new premise to the discussion about the sources of uncertainty of RNAi results.

shRNAs targeting mouseAdam10diminish cell response to proinflammatory stimuli independently ofAdam10silencing

“…S4 " type="url"/> ). We also compared the results of RNAseq analyses of unstimulated and cytokine-stimulated MC38CEA cells, in which ADAM17 expression was silenced by shRNAs or CRISPR/Cas9 (GEO repository accession numbers: GSE181084 and GSE181271, respectively) ( Czarnek and Bereta, 2021a , b ). The analysis did not reveal common inflammation-related genes affected by diminished ADAM17 expression, independently of the method of its silencing.…”

“…MC38CEA cells express only few proinflammatory proteins (GEO repository: GSE181084 or GSE181271) ( Czarnek and Bereta, 2021a , b ). We chose CCL2 (C-C chemokine ligand 2), known also as MCP1 (macrophage chemoattractant protein 1) and CCL7 (MCP3) to examine whether the influence of shRNAs was limited to iNOS expression or is a more general phenomenon.…”

“…. Czarnek and Bereta (2021a , b) . RNA seq analysis of MC38CEA cells with diminished ADAM17 expression due to Crispr/Cas9 gene editing or control cells.…”

“… Data availability Datasets deposited in NCBI Gene Expression Omnibus (GEO) database: Czarnek and Bereta (2021a , b) . RNA seq analysis of MC38CEA cells transduced with pLKO1 vectors encoding shRNAs targeting Adam17 or control shRNA.…”