The specificity of CCRS regulating target genes expression

In this study, we fused CasRx crRNA with antisense ribozyme to construct a synthetic fusion guide RNA that can interact with both CasRx protein and ribozyme, and tested the ability of this approach in RNA knockdown and cancer gene therapy. We show that the CasRx-crRNA-ribozyme system (CCRS) is more efficient for RNA knockdown of mRNAs and non-coding RNAs than conventional methods including CasRx, shRNA, and ribozyme. In particular, CCRS was more effective than wild-type CasRx when targeting multiple transcripts simultaneously. We next used bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes in vitro and in vivo. CCRS showed a higher anticancer effect than conventional methods, consistent with the gene knockdown results. Thus, our study demonstrates that CCRS expands the design ideas and RNA knockdown capabilities of Cas13 technology, and has the potential to be used in disease treatment.