Harnessing multiplexed Cas9 genome editing for sequential genetic manipulations and recording activities in cell lineages

总结 对于生物学家来说，时间是一个问题，特别是对于那些对癌症，干细胞和发育感兴趣的人来说， 生物学尽管细胞过程发生在真实的时间中，但我们通常试图通过拼凑来理解生物学。 结合静态终点实验获得的信息。这个提案的目的是扩大一部小说 技术，以提供创新的处理问题所提出的时间在生物学。该技术记录了 使用CRISPR/Cas9基因编辑工具记录细胞自身基因组内的事件， 是为了在细胞中执行类似机器的指令而开发的分类账可以被下一代DNA“读取” 排序，允许一次阅读许多分类账条目。多个分类账的存在 连续DNA链上的条目使来自许多细胞的所有条目能够在单个细胞上被读取 水平记录系统是基于将多个模块活动串联起来， 通过顺序激活的步骤级联过程;每个步骤由单个模块编码。我们有 开发和验证了各个模块的基本组件，在这里，我们建议建立和 优化以预先编程的顺序连接在一起的一系列模块。拟议的研究由以下人员组织 三个具体目的：1）用10-模块长级联扩展干细胞谱系追踪的持续时间， 2)使用我们的技术记录谱系中单个细胞中Wnt信号传导的历史，以及3）控制 细胞记录的棘轮进展，使得细胞事件可以用细胞分裂来识别 号码追踪器该提案的完成将为生物学家提供大量新的能力， 询问生物系统的功能。

SUMMARY Time is a problem for biologists, especially for those interested in cancer, stem cell, and developmental biology. Whereas cellular processes occur in real time, we most often try to understand biology by piecing together information obtained with static end-point experiments. The goal of this proposal is to expand a novel technology to provide innovative of dealing with problems posed by time in biology. The technology records a ledger of events within the cell’s own genome with CRISPR/Cas9 gene editing tools, which have been developed to execute machine-like instructions in cells. The ledger can be “read” by next generation DNA sequencing, allowing the reading many ledger entries at one time at once. The presence of multiple ledger entries on a contiguous DNA strand enables all the entries from many cells to be read at the individual cell level. The system of recording is based on combining multiple modular activities in series to develop a cascading process through sequentially activated steps; each step is encoded by a single module. We have developed and validated the essential components of individual modules, and here we propose to build and optimize series of modules linked together in pre-programmed orders. The proposed research is organized by three Specific Aims of 1) expanding the duration of stem cell lineage tracking with a 10-module long cascade, 2) record the history of Wnt signaling in individual cells in a lineage using our technology, and 3) control a ratcheting progression of cellular recording such that cellular events can be identified with a cell division number tracker. Completion of the proposal will provide a substantial new capability for biologists in interrogating the functions of biological systems.