Shenzhen Institute of Translational Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Health Science Center, Shenzhen University, Shenzhen, 518035, China.
Department of Urology, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou, 450052, China.
Genome Biol. 2023 Jan 17;24(1):9. doi: 10.1186/s13059-023-02852-w.
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.
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.
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.
CRISPR-Cas13 是一种新兴的 RNA 敲低技术，可与 RNAi 相媲美。在所有 Cas13 成员中，CasRx 可高精度、高效地在人细胞中降解 RNA。然而，目前尚不清楚该技术的效率是否可以进一步提高，并应用于基因治疗。
在这项研究中，我们将 CasRx crRNA 与反义核酶融合，构建了一种能够与 CasRx 蛋白和核酶相互作用的合成融合向导 RNA，并测试了该方法在 RNA 敲低和癌症基因治疗中的能力。我们表明，CasRx-crRNA-核酶系统 (CCRS) 在 mRNA 和非编码 RNA 的 RNA 敲低方面比包括 CasRx、shRNA 和核酶在内的常规方法更有效。特别是，CCRS 在同时靶向多个转录本时比野生型 CasRx 更有效。接下来，我们使用膀胱癌作为模型，在体外和体内评估 CCRS 靶向多个基因的抗癌效果。CCRS 显示出比常规方法更高的抗癌效果，与基因敲低结果一致。
因此，我们的研究表明，CCRS 扩展了 Cas13 技术的设计思路和 RNA 敲低能力，具有在疾病治疗中应用的潜力。
