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bioRxiv · 10.1101/2020.11.25.398354

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

Czarnek, M.·Sarad, K.·Karas, A.·Kochan, J.·Bereta, J.
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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(R) 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.

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Czarnek, M., Sarad, K., Karas, A., Kochan, J., Bereta, J.. 2020-11-26. Non-targeting control for MISSION(R) shRNA library silences SNRPD3 leading to cell death or permanent growth arrest. https://doi.org/10.1101/2020.11.25.398354

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