Yadong Wang,1,* Jinjin Zhu,1,* Shangwen Liu,1 Zhengbo Sun,1,2 Guibiao Wen,1 Dakun Huang,1 Mianxiong Chen,1 Yuchen Liu,3 Feng Lin1 

1Department of Surgery, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical School of Guangzhou University of Chinese Medicine, Shenzhen, People’s Republic of China; 2Materials and Interfaces Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, People’s Republic of China; 3Guangdong Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Feng Lin; Zhengbo Sun, Department of Surgery, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical School of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian, Shenzhen, Guangdong, 518033, People’s Republic of China, Email lf0237@gzucm.edu.cn; Sunzhengbo@whu.edu.cn

Introduction: The FGFR3-TACC3 fusion gene exists in a variety of malignant tumors, including bladder cancer. In our ongoing research on the CRISPR-Cas13a gene-editing system, we reported the use of CRISPR-Cas13a gene-editing system to knockout FGFR3-TACC3 and inhibit the proliferation of bladder tumor cells.
Purpose:  This study aimed to use the CRISPR-Cas13a gene-editing system to target the FGFR3-TACC3 fusion gene in bladder cancer cells, which has the potential to be a new and effective treatment for bladder cancer.
Materials and Methods: The efficacy of the CRISPR-Cas13a gene-editing system was analysed by qRT-PCR. The inhibitory effects of Cas13a-mediated knockdown of the FGFR3-TACC3 fusion gene on the proliferation of RT4 and RT112 cell lines were assessed utilizing CCK-8, EdU, and organoid formation assays. Subsequently, the comparative tumorigenic capability of RT4 cells with FGFR3-TACC3 knockdown achieved by Cas13a was examined in a nude mouse model.
Results: At the cellular level, the comparative analysis of FGFR3-TACC3 knockdown efficacy between CRISPR-Cas13a and shRNA revealed a more pronounced reduction with the former. This knockdown effectively curtailed cellular proliferation, with CRISPR-Cas13a-mediated knockdown exhibiting a superior inhibitory effect over shRNA-mediated knockdown. In organoid cultures derived from RT4 cells, a similar trend was observed, with Cas13a-mediated knockdown of FGFR3-TACC3 leading to a more substantial suppression of proliferation compared to shRNA-mediated knockdown. In vivo tumor models corroborated these findings, demonstrating a significantly diminished tumor volume in the Cas13a-treated cohort relative to both the control and shRNA-treated groups.
Conclusion: The CRISPR-Cas13a gene-editing system has been demonstrated to significantly suppress tumor proliferation both in vitro and in vivo, thereby presenting itself as a promising candidate for a novel and efficacious therapeutic intervention in bladder cancer treatment.

Keywords: FGFR3-TACC3, oncogenic mutation, CRISPR-Cas13a, mRNA knockdown, bladder cancer