Abstract
Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs — very long intergenic non-coding (vlinc) RNAs — in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.
Highlights
The lncRNA class of transcripts dominates the transcriptional output of a mammalian genome[1,2,3] and as such attracted vast research interest[4,5]
We used a co-transfection assay where two plasmids constitutively expressing (1) nuclear-localized Cas13-msfGFP fusion protein and (2) a specific gRNA are electroporated into K562 chronic myeloid leukemia (CML) cells followed by assessing the depletion of the target transcript after 24 h using RT-qPCR
We used a positive control gRNA against protein-coding mRNA KRAS employed in the original study describing the CRISPR/Cas[13] system[22] and a gRNA targeting Gaussia luciferase (Gluc) from the same source as the non-specific control
Summary
The lncRNA class of transcripts dominates the transcriptional output of a mammalian genome[1,2,3] and as such attracted vast research interest[4,5]. In this work, we have explored a possibility of applying CRISPR/Cas[13] system for high-throughput phenotypic screens for lncRNAs and mRNAs in a context of a stable expression system In this design, each cell stably expresses a specific gRNA that upon induction of Cas[13] integrated into the genome can induce knockdown of the target transcript. We have built in several layers of controls to allow for as accurate measurement of a transcript-related effect on cell survival as currently achievable To test this system, we have chosen a recently-discovered class of very long intergenic non-coding (vlinc)RNAs that represent nuclear polyA− transcripts of over 50 kb widespread in a mammalian genome[23,24]. We provide evidence strongly suggesting that majority of the tested vlincRNAs represent functional RNA species
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