Abstract 1094: Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma

Abstract Background: CRISPR-Cas13 (Cas13) is an RNA editing system that can knockdown gene transcripts with higher efficiency and specificity than RNA interference technologies. We are exploring the utility of Cas13 as a robust novel functional genomics tool to understand the biology of the PAX3-FOXO1(P3F1) gene fusion in alveolar rhabdomyosarcoma (ARMS). Our goal is to identify gene dependencies and novel druggable targets. [LB1] Aim: This study aims to functionally characterize P3F1 in paediatric cancer cell lines using Cas13. [LB2] Methods & Results: Transient transfection and viral transduction of Cas13 vector systems were used to demonstrate that Cas13 knockdown of target gene expression can be achieved in ARMS cell lines. We developed guides to specifically target P3F1 expression and tested their efficiency in HEK293T cells. Cas13 function was validated with a mCherry knockdown proof-of-concept expression in the ARMS cell lines Rh5 and Rh30. 90-95% knockdown efficiency was seen over three independent experiments. Ten P3F1 breakpoint targeting guides were designed and screened first in a HEK293T P3F1 overexpression model to select optimal high efficiency targeting guides. The highest efficiency targeting guide showed a GFP reduction of 70% in three independent experiments. High-efficiency Cas13 guides were taken forward into Rh5 cells to knockdown endogenous P3F1 expression which were validated by western blotting and RT-PCR. Western blots of Cas13 sorted Rh5 cells showed that guide 1150 was able to knockdown P3F1 protein expression by ~70%. Discussion/Conclusion: These findings demonstrate the high efficiency and specificity of the Cas13 system to silence P3F1, and other oncogenic fusion drivers. Further studies will utilize Cas13 to characterize the molecular and cellular consequences of P3F1 gene-silencing in patient-derived ARMS cell lines. Citation Format: Andrea Zhao, Lauren M. Brown, Mohamed Fareh, Paul G. Ekert. Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1094.