Personalised medicine initiatives such as the Zero Childhood Cancer Program have highlighted the genomic heterogeneity of paediatric cancers, and the value of molecular characterisation to deliver more effective treatments to patients. However, as more novel variants are identified, there is both a need and an opportunity to characterise their functions and therapeutic targetability. To do this, we are developing a comprehensive library of CRISPR/Cas-engineered isogenic paediatric cancer cell lines in which we can manipulate gene expression through knockout (CRISPR/Cas9), knockdown (CRISPR/Cas13), or overexpression (CRISPRa) strategies. We also utilise a nuclease-dead variant of Cas13d (dCas13d) as a control. Leveraging CRISPR/Cas technology, we aim to provide tools for the functional characterisation of candidate driver genes and novel variants identified in paediatric malignancies.
To date, we have successfully generated 22 CRISPR/Cas cell lines, encompassing a wide range of paediatric cancers including neuroblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma. Across the CRISPR/Cas13d cell lines generated we see variable expression of doxycycline (dox)-induced Cas13d. Additionally, clonogenic assays show that upon dox-induced expression of Cas13d there is a dose-dependent reduction in colony forming capacity, independent of guide expression. Notably, no significant changes were observed in cells expressing either dCas13d or Cas9, which highlights the inherent toxicity of Cas13d. Moreover, our findings suggest that guide expression levels may be an important limiting factor in the efficiency of gene silencing, indicating that both guide and Cas13d expression need to be carefully controlled. Further exploration of strategies to mitigate Cas13d toxicity while optimising guide expression to enhance gene silencing efficacy are underway.
Alongside this, we are utilising a unique pipeline we have developed based on CRISPR/Cas9 technology to generate PDGFRA structural variants by targeting intronic regions adjacent to the deleted exons. This approach enables us to recreate these PDGFRA variants endogenously and further characterise their function.
In summary, the cell lines developed in this project offer a valuable resource for investigating the molecular mechanisms driving paediatric cancers. Furthermore, they serve as a toolkit for identifying and exploring potential therapeutic vulnerabilities, advancing our understanding of cancer biology and contributing to the development of more targeted, effective treatments for paediatric patients.