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bioRxiv · 10.1101/2022.08.30.505797

Chemically synthesized guide RNAs can direct CRISPR-CasRx cleavage of circRNAs with high efficiency and specificity

Rahimi, K.·Andersen, M. S.·Seeler, S.·Hansen, T. B.·Kjems, J.
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Abstract

Circular RNAs (circRNAs) are characterized by a covalently closed circular structure, formed from pre-mRNAs through an alternative splicing mechanism named back-splicing. CircRNAs have been shown to play a regulatory role in the development of eukaryotic organisms and to be implicated in human diseases. However, the extensive sequence-overlap between circRNAs and their linear RNA counterparts makes it technically difficult to deplete circRNAs without affecting their linear host, which complicates functional studies. Therefore, it is important to identify the most efficient and specific strategy for circRNA depletion. In this study, we demonstrate that CRISPR/RfxCas13d (CasRx)-mediated circRNA depletion is, for the circRNAs studied, more efficient than Argonaute 2-dependent short hairpin RNA (agoshRNA)-mediated depletion and with fewer off-target effects on the linear host RNAs. Furthermore, we show that synthetic guide RNAs (syn-gRNAs) can be used in combination with CasRx to efficiently deplete circRNA, ciRS-7. Finally, none of the knockdown (KD) strategies tested (pre-gRNA, gRNA, syn-gRNA and agoshRNA) showed any significant off-target effects on the global transcriptome. Taken together, CasRx-mediated circRNA KD strategies, using either vector-based or syn-gRNA, are useful tools for future studies on circRNA functions.

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Rahimi, K., Andersen, M. S., Seeler, S., Hansen, T. B., Kjems, J.. 2022-08-30. Chemically synthesized guide RNAs can direct CRISPR-CasRx cleavage of circRNAs with high efficiency and specificity. https://doi.org/10.1101/2022.08.30.505797

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