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 (RNAi) techniques, accumulating evidence indicates that RNAi analyses might be seriously biased due to the off-target effects of gene-specific short hairpin RNAs (shRNAs). Our findings indicated that off-target effects of non-targeting shRNA comprise another source of misinterpreted shRNA-based data. We found that SHC016, which is one of two non-targeting shRNA controls for the MISSION (commercialized TRC) library, exerts deleterious effects that lead to elimination of the shRNA-coding cassette from the genomes of cultured murine and human cells. Here, we used a lentiviral vector with inducible SHC016 expression to confirm that this shRNA induces apoptosis in murine cells and senescence or mitotic catastrophe depending on the p53 status in human tumor cells. We identified the core spliceosomal protein, small nuclear ribonucleoprotein Sm D3 (SNRPD3), as a major SHC016 target in several cell lines and confirmed that CRISPRi knockdown of SNRPD3 mimics the effects of SHC016 expression in A549 and U251 cells. The overexpression of SNRPD3 rescued U251 cells from SHC016-induced mitotic catastrophe. Our findings disqualified non-targeting SHC016 shRNA and added a new premise to the discussion about the sources of uncertainty in RNAi results.

shRNAs targeting mouseAdam10diminish cell response to proinflammatory stimuli independently ofAdam10silencing

“…They demonstrated how erroneous RNAi data led to selection of incorrect targets for cancer therapy and even to initiation of clinical trials, which were bound to fail. In our previous work we reported that also control shRNA, which by definition should not influence expression of any gene, might elicit powerful off-target effects that invalidate the reliable interpretation of RNAi data ( Czarnek et al, 2021 ). Thus, in the case of shRNAs application, off-target effects seem to be a common phenomenon.…”

“…Oligonucleotides encoding the shRNA sequences ( ) were annealed in 1×Taq buffer and ligated into linearized plasmids. Lentiviral vectors were produced as described previously ( Czarnek et al, 2021 ).…”

The homeostatic function of Regnase‐2 restricts neuroinflammation

“…The pSBbi‐Pur‐dCas9‐KRAB‐meCP2‐hU6‐sgRNA‐SapI plasmid, used for Reg‐2 knockdown was a gift from Maria Czarnek 45 . sgRNA sequence targeting Zc3h12b was designed using the CHOPCHOP tool 46 .…”

Tumor suppressive functions of WNT5A in rhabdomyosarcoma

“…In all settings and if not stated otherwise, RMS cells transduced with either the empty pBABE or the empty pGIPZ vectors served as respective controls [CTR; please note that it was decided to use an empty vector for the WNT5A shRNA validation experiments, because an empty vector was also used for the WNT5A overexpression experiments. In addition, empty vectors are frequently used in knockdown experiments ( 27 , 28 )]. pBABE and GIPZ plasmids both contain a puromycin resistance cassette.…”