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.
Aim: To functionally characterise P3F1 by utilising Cas13 mediated silencing of the endogenous fusion oncogene in paediatric cancer cell lines.
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. Cas13 function was validated with an mCherry knockdown proof-of-concept experiment in the ARMS cell lines Rh5 and Rh30. 90-95% knockdown efficiency was seen over three independent experiments. We then designed ten Cas13 guides that span across the breakpoint of P3F1 and screened the guides in a HEK293T P3F1 overexpression model to select optimal high efficiency targeting guides. The highest efficiency targeting guide induced a GFP reduction of 70% in three independent experiments. High-efficiency Cas13 guides were then tested in Rh5 cells to knockdown endogenous P3F1, which were validated by Western blotting and RT-PCR. Western blot analysis of Cas13 sorted Rh5 cells showed that one P3F1 targeting guide was able to knockdown P3F1 protein expression by ~70%.
Discussion/Conclusion: These findings demonstrate the high efficiency of the Cas13 system to silence P3F1, and other oncogenic fusion drivers. Further studies will utilise Cas13 to characterise the molecular and cellular consequences of P3F1 gene-silencing in patient-derived ARMS cell lines.