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bioRxiv · 10.1101/2025.11.27.690751

Off-target effects as confounders of Cas13d-based lncRNA screens

Montero, J. J.·Trozzo, R.·Rad, R.
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Abstract

Despite their recognized role in biology, a majority of the [~]100,000 lncRNA genes remain functionally uncharacterized. In a recent study (Liang WW et al., Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells, Cell, 2024), Liang et al. utilized the RNA nuclease Cas13d to perturb [~]6,200 lncRNAs in fitness screens across five cell lines - thereby identifying 778 lncRNAs with broad or context-specific essentiality. However, previous screens reported a lower proportion of essential lncRNAs. To investigate this discrepancy, we re-analysed Liang et al.s data and found that 68.1% of gRNAs causing fitness defects have off-targets in essential protein-coding genes. This caused numerous false-positive hits, particularly among lncRNAs classified as broadly essential. Off-target effects also compromise the studys validation efforts, including experiments combining single-cell transcriptomics and lncRNA-perturbations, which confirm the downregulation of off-target protein-coding genes identified in our analyses. The large number of false-positive hits reported by Liang et al. undermines the studys biological conclusions and endangers future research building on these data, if not considered.

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Montero, J. J., Trozzo, R., Rad, R.. 2025-11-29. Off-target effects as confounders of Cas13d-based lncRNA screens. https://doi.org/10.1101/2025.11.27.690751

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