Cas13 is a new reprogrammable CRISPR effector that targets single-stranded RNAs with high efficiency and specificity. This bacterial nucleoprotein holds a great potential to suppress pathogenic transcripts with single-base precision. For instance, we recently demonstrated efficient suppression of replication-competent SARS-CoV-2 variants with reprogrammed Cas13b1. However, the poor understanding of the molecular bases that govern Cas13 targeting restricts its use for broader applications.
To uncover the molecular bases of Cas13b-mediated target recognition and cleavage processes, we developed innovative library screens in mammalian cells where we designed >200 tiled CRISPR RNAs (crRNAs) targeting several transcripts of interest with single-base resolution. We built in-house bioinformatic analysis pipelines to comprehensively investigate hidden parameters that govern Cas13 activity in these datasets. We found that, unlike Cas9 and other CRISPR systems, Cas13b is not constrained by any protospacer-flanking sites (or PAM-like), highlighting its design flexibility. Remarkably, we revealed previously unknown RNA motifs within the spacer sequence of crRNAs that are either highly enriched or depleted in extremely potent and unproductive crRNAs, respectively. Among these RNA motifs, we found a unique sequence at the 5’ end of the spacer that greatly enhances Cas13b potency. To further validate these findings, we designed de novo crRNAs harbouring the unnatural RNA motifs that we identified in the screen, which exhibited maximum potency that largely outperformed conventionally designed crRNAs.
Finally, we leveraged these molecular features to reprogram Cas13b to silence several gene fusion transcripts that drive a variety of tumours. We show that targeting the breakpoint of fusion transcripts with de novo designed tiled crRNAs yields very high and specific silencing, with absolute discrimination between tumour-associated fusion transcripts and the wild-type variants expressed in normal cells (unpublished).
Taken together, this study revealed key molecular features for efficient Cas13b reprogramming and provides a molecular blueprint for its deployment against pathogenic transcripts