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

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.</p><p dir="ltr">In this study, we introduce two complementary methods to elucidate the molecular mechanism of Cas13-mediated RNA cleavage.

First, we adapted a high-throughput <i>in vitro</i> cleavage assay to identify cleavage motifs across diverse Cas13 proteins. Second, we developed an <i>in vivo</i> adaptor-ligation approach that maps Cas13 cleavage sites at single-nucleotide resolution. By integrating <i>in vitro</i> and <i>in vivo</i> datasets, we built a robust model of Cas13-mediated target RNA cleavage.</p><p dir="ltr">Firstly, we proposed an RNA foldback model where the target RNA needs to be folded back to the active site for cleavage.