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

项目摘要 揭示细胞之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，并揭示了导致发育复杂性的过程。 有机体然而，目前在癌症生物学和发育生物学中研究这些关系是困难的。 受限于体内细胞谱系追踪和重建的困难。细胞谱系的完全重建 需要非侵入性的活细胞成像，其限于相对少量的细胞并且对于 生理相关的研究。尽管最近用于CRISPR介导的基因组条形码化的系统 作为一个有前途的替代品出现，他们遭受的多样性和血统的产生率的限制- 识别使它们不适合克隆进化和谱系发育研究的信息， 尤其是在癌症中。此外，这些方法不能明确地捕获细胞分裂，细胞分裂的基本参数。 体细胞进化可能会影响我们对癌症对干预反应的理解。 为了改善癌症模型中群体动态和细胞谱系信息的恢复，我们提出了一种细胞- 周期诱导的遗传构建体，其将在单个位点中记录每个周期的分裂和谱系的序列。 cell.我们的方法使用细胞周期调节的泛素化来控制无供体的基于CRISPR的 基因组记录装置，其在每次分裂时对细胞进行条形码化。在这项研究中，我们将评估 我们的设备用于跟踪克隆人类和小鼠细胞群体中的谱系信息和细胞分裂动力学。 为了证明其在发育研究中的研究潜力，我们将联合收割机结合我们的细胞分裂条形码技术， 利用单细胞RNA测序确定癌症和hiPSC中健康和异常分化的策略 衍生类器官。如果成功，我们对细胞分裂、克隆性和谱系恢复的改善将得到改善。 我们对发展的理解，解释癌症耐药性的进化来源，并指导医生 转化研究中更有效的治疗方法。

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.