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

细胞转化的评估（即在形态、生长控制或功能上获得恶性特征）已被证明在体外模型中存在问题。软琼脂细胞转化试验已被验证，但仅在有限数量的永生化或混合群体胚胎细胞和特异性的测定受到质疑。此外，该模型中细胞转化的遗传基础尚未完全描述。使用来自单个人类干细胞的遗传和表型稳定的类器官应该为细胞增殖和转化提供更好的模型。类器官具有3D形态的优势，能够从几个主要器官中提取，并且可以通过引入细胞类型特异性ki67荧光蛋白来标记来展示增殖效应。类器官的稳定性也依赖于几种生长因子和抑制剂，如EGF、Wnt、BMP抑制剂、TGFb抑制剂。这些因子是已知的与癌症发生有关的途径，在退出这些因子后的生长将指示细胞表型和/或转化性变化。研究人员还提出，该模型可用于研究CRISPR/Cas9基因组编辑的致癌性机制以及潜在的脱靶效应，目前尚不需要进行体外试验来评估对癌基因不当编辑的担忧。该项目将从各种组织来源中开发类器官，包括带有ki67标签的类器官，以及评估在参考致癌物治疗后停止生长因子，以及在转染特异性和混杂的指导RNA和相关Cas9蛋白后的增殖和形态学变化。比较数据将从其他体外方法中产生，以分析细胞转化，例如软琼脂试验。参考致癌物将在该试验中与CRISPR/Cas9编辑诱导细胞转化的潜力一起进行分析（此前尚未评估）。该学生还将使用下一代测序技术来识别细胞和类器官培养中涉及形态变化的特定基因。成功将为理解和评估形态学变化和癌变提供新的和更相关的模型。

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.