A cell-cycle induced genetic recorder for simultaneous recovery of cell divisions and lineage

项目摘要 揭示细胞间的谱系关系有可能说明肿瘤发生的进化模式。 以及对治疗的抵抗力，并阐明了导致发育复杂的过程 有机体。然而，在癌症生物学和发育生物学中研究这些关系目前正在进行 受体内细胞谱系追踪和重建困难的限制。细胞谱系的完整重建 需要非侵入性活细胞成像，这仅限于相对较少的细胞数量，对于 与生理相关的研究。尽管最近用于CRISPR介导的基因组条码的系统已经 作为一种有希望的替代选择，它们在血统的多样性和世代率方面受到限制- 识别使它们不适合克隆进化和谱系发展研究的信息， 尤其是在癌症方面。此外，这些方法无法显式捕获细胞分裂，这是 这可能会影响我们对癌症对干预的反应的理解。 为了提高从癌症模型中恢复种群动态和细胞谱系信息，我们提出了一种细胞-- 循环可诱导的遗传结构，将在单个基因座上记录每个基因的分裂和谱系序列 手机。我们的方法使用细胞周期调节的泛素化来控制基于CRISPR的无供体的活性 基因组记录设备，在每个分裂时对细胞进行条形码编码。在这项研究中，我们将评估 我们的设备跟踪人类和小鼠克隆细胞群体的谱系信息和细胞分裂动力学。 为了展示它在发育研究中的研究潜力，我们将结合我们的细胞分裂条形码 用单细胞RNA测序识别癌症和HiPSC健康和异常分化的策略 衍生的有机化合物。如果成功，我们对细胞分裂、克隆和血统恢复的改善将会改善 我们对发育的理解，解释癌症耐药性的进化来源，并指导从业者 在转化性研究中走向更有效的治疗方法。

Project Summary Revealing the lineage relations among cells has the potential to illustrate evolutionary patterns in tumor initiation and resistance to therapies and shed light on the processes which give rise to developmentally complex organisms. However, studying these relationships in cancer biology and developmental biology is currently limited by the difficulty of cell lineage tracing and reconstruction in-vivo. Complete reconstructions of cell lineages require non-invasive live cell imaging, which is restricted to relatively small numbers of cells and impractical for physiologically-relevant studies. Although recent systems for CRISPR-mediated genome barcoding have emerged as a promising alternative, they suffer limitations in the diversity and generation rate of lineage- identifying information which render them unsuited to studies of clonal evolution and lineage development, especially in cancer. Moreover, these methods fail to explicitly capture cell divisions, a basic parameter of somatic evolution which could impact our understanding of a cancer's response to intervention. To improve recovery of population dynamics and cell lineage information from cancer models, we propose a cell- cycle inducible genetic construct that will record, in a single locus, the sequence of divisions and lineage of each cell. Our approach uses cell-cycle regulated ubiquitination to control the activity of a donor-free CRISPR-based genome recording device which barcodes cells upon each division. In this study, we will evaluate the ability of our device to track lineage information and cell division dynamics in clonal human and mouse cell populations. To demonstrate its investigative potential in developmental studies, we will combine our cell-division barcoding strategy with single-cell RNA sequencing to identify healthy and abnormal differentiation in cancer- and hiPSC- derived organoids. If successful, our improvements to recovery of cell division, clonality, and lineage will improve our understanding of development, explain evolutionary sources of resistance in cancer, and guide practitioners toward more effective treatments in translational research.