Supporting data for Mechanistic insights into Cas13 cleavage and the development of RNA segment editing

Abstract

Since its discovery in 2016, the RNA-targeting CRISPR-Cas13 protein is widely adopted for precise knockdown of target RNAs in human cells. However, conflicting reports from different groups over the years raised questions about the specificity of Cas13 knockdown. The ambiguity largely stems from the fact that the exact cleavage sites on target RNAs remain poorly defined due to current limitations in structural and biochemical approaches.In this study, we introduce two complementary methods to elucidate the molecular mechanism of Cas13-mediated RNA cleavage. First, we adapted a high-throughput in vitro cleavage assay to identify cleavage motifs across diverse Cas13 proteins. Second, we developed an in vivo adaptor-ligation approach that maps Cas13 cleavage sites at single-nucleotide resolution. By integrating in vitro and in vivo datasets, we built a robust model of Cas13-mediated target RNA cleavage.Firstly, we proposed an RNA foldback model where the target RNA needs to be folded back to the active site for cleavage. Secondly, we found that certain Cas13 proteins predominantly cleave positions proximal to the binding site. We further demonstrate that cleavage-site specificity can be enhanced by engineering the HEPN catalytic centre. Finally, we repurposed the cleavage-site mapping method for RNA segment editing (RSE), enabling the removal or replacement of large regions of endogenous RNA in cells. To our knowledge, this represents the first demonstration of Cas13-based RNA segment editing based on targeted cleavage and repair.