Use of organoid cultures to assess genome editing technology and model human physiology

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Assessment of cell transformation (i.e. the acquisition of malignant characteristics in morphology, growth control orfunction) has proven problematic to model in vitro. The soft agar cell transformation assay has been validated, but only in alimited number of immortalised or mixed population embryonic cells and the specificity of the assay has been questioned.Furthermore, the genetic basis of cell transformation in this model has not been fully delineated. The use of genetically andphenotypically stable organoids derived from single human stem cells should provide a better model for cell proliferation andtransformation.Organoids have the advantage of 3D morphology, being able to be derived from several major organs and can be engineeredto demonstrate proliferative effects by introducing a cell type specific ki67 fluorescent protein to tag. Organoid stability isalso dependent on several growth factors and inhibitors e.g. EGF, Wnt, BMP inhibitor, TGFb inhibitor. Growth followingwithdrawal of these factors, which are known pathways implicated in cancer genesis, will be indicative of cell phenotypicand/or transformative changes. It is also proposed that this model could be used to investigate mechanisms of prooncogenicityand also the potential off-target effect of genome editing by CRISPR/Cas9, for which there is currently an unmetneed for in vitro assays to assess concerns around inappropriate editing in oncogenes.The project will develop organoids from a variety of tissue sources, including organoids with the ki67 tag, and assessment ofproliferative and morphological changes following withdrawal of growth factors after treatment with reference carcinogensand following transfection with specific and promiscuous guide RNA and associated Cas9 protein. Comparative data will begenerated from alternative in vitro methodologies to analyse cell transformation e.g. the soft agar assay. Referencecarcinogens will be analysed in this assay along with the potential of CRISPR/Cas9 editing to induce cell transformation(which has not been previously assessed). The student will also use next generation sequencing technologies to identifyspecific genes involved in morphological changes in both cell and organoid cultures. Success will provide new and morerelevant model for understanding and assessment of morphological changes and carcinogenesis.