Deep cell history tracking: engineering cells that write their detailed life stories into their DNA to study DNA damage

项目摘要 DNA记录是一项最近发展起来的技术，它可以跟踪感兴趣的细胞信号事件 随着时间的推移，不会将细胞从其生理环境中移除。在DNA记录实验中，从基因上讲 表达DNA记录器的工程细胞由于瞬时突变而获得了“记录位点”上的突变 细胞事件，而不干扰它们的正常表型。在实验结束时，一旦他们最终 表型是已知的，可以收集细胞，并通过对记录基因进行测序来确定它们的历史。 这里提出的工作将导致一种DNA记录器，它不像目前的技术那样局限于记录一个 或每个实验两个细胞事件，但可以并行记录数十个事件，允许不偏不倚 在任何与基因工程细胞兼容的环境中检查细胞历史。在这个项目中，新的 DNA记录仪将被用来研究经历显著自发DNA的细胞的长期影响 损坏。尽管对dna损伤的研究非常深入，但以前还不可能追踪到稀有细胞。 经历显著的自发损伤，特别是在活体哺乳动物的环境中。新的DNA 记录器将分阶段发展：第一，细胞内记录机，在现有记录器的基础上， 将为与DNA损伤反应相关的少量细胞事件创建。其次，忠诚度 这一新的记录机制将在培养细胞和癌症异种移植中得到广泛验证。最后，一个 将开发和验证新的记录体系结构，然后将其引入转基因小鼠以 确定在正常发育过程中，自发的DNA损伤如何影响细胞命运的决定。

Project Summary DNA recording is a recently developed technology that allows cellular signaling events of interest to be followed over time without removing cells from their physiological context. During a DNA recording experiment, genetically engineered cells expressing the DNA recorder acquire mutations at a “recording locus” as the result of transient cellular events, without perturbing their normal phenotype. At the end of the experiment, once their eventual phenotype is known, cells can be collected and their histories determined by sequencing the recording locus. The work proposed here will lead to a DNA recorder that is not limited, as current technology is, to recording one or two cellular events per experiment, but can record dozens of events in parallel, allowing an unbiased examination of cell history in any context compatible with genetically engineered cells. In this project, the new DNA recorder will be used to study long-term effects for a cell of experiencing significant spontaneous DNA damage. Although DNA damage is very well-studied, it has not previously been possible to follow the rare cells that experience significant spontaneous damage, especially in an in vivo mammalian context. The new DNA recorder will be developed in stages: First, intracellular recording machinery, based on a pre-existing recorder, will be created for a small number of cellular events associated with the DNA damage response. Next, the fidelity of this new recording machinery will be extensively validated in cultured cells and cancer xenografts. Finally, a new recording architecture will be developed and validated, before being introduced into a transgenic mouse to identify how spontaneous DNA damage affects cell fate determination during normal development.