Enhanced RNA knockdown efficiency with engineered fusion guide RNAs that function with both CRISPR-CasRx and hammerhead ribozyme
Yonghao Zhan, Congcong Cao, Aolin Li, Hongbing Mei, Yuchen Liu
Abstract
BACKGROUND: CRISPR-Cas13 is a newly emerging RNA knockdown technology that is comparable to RNAi. Among all members of Cas13, CasRx degrades RNA in human cells with high precision and effectiveness. However, it remains unclear whether the efficiency of this technology can be further improved and applied to gene therapy. RESULTS: In this study, we fuse CasRx crRNA with an 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 is more effective than wild-type CasRx when targeting multiple transcripts simultaneously. We next use bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes in vitro and in vivo. CCRS shows a higher anticancer effect than conventional methods, consistent with the gene knockdown results. CONCLUSIONS: 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.