Single-nucleotide-resolution mapping of RNA-targeting Cas system cleavage sites

Abstract

DNA-targeting CRISPR-Cas systems have been the primary genome editing tool in recent years. RNA-targeting CRISPR-Cas systems, however, have garnered blooming research interest due to the possibility of temporary transcriptome engineering instead of permanent genome alterations. The recently discovered RfxCas13d (or CasRx) and SthCsm systems are active in human cells and have been applied in different applications such as repeat-expansion disease therapies, RNA imaging, RNA editing, and so on. Be that as it may, the precise biochemical mechanisms of these systems remain elusive and hinder the further development of different RNA interrogation tools. In this study, we applied advanced techniques to examine the cleavage sites of CasRx in vitro and in vivo. With the bind-and-wash approach, we were able to enrich the cis-cleavage events of CasRx in vitro and further dissect its detailed biochemical activities. With the next-generation sequencing approach termed 5 ́OH-seq, we revealed the in vivo cleavage sites of CasRx. As a result, this work provides detailed guidelines for the research community when applying these systems. We also envisioned the development of new methods or tools based on the new-found knowledge in the future.