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

摘要 时间对生物学家来说是个问题，尤其是对癌症、干细胞和发育感兴趣的人。 生物学。虽然细胞过程是实时发生的，但我们最常试图通过拼接来理解生物学 结合静态终点实验获得的信息。这项提议的目的是扩展一部小说 技术为处理生物学中的时间问题提供了创新。这项技术记录了 使用CRISPR/Cas9基因编辑工具对细胞自身基因组内的事件进行分类，这些工具已经被 开发用于在单元中执行类似机器的指令。账本可以被下一代DNA“读懂” 排序，允许一次读取多个分类帐分录。存在多个分类帐 连续DNA链上的条目使来自多个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开发的 通过顺序激活的步骤进行级联处理；每个步骤由单个模块编码。我们有 开发并验证了各个模块的基本组件，在此我们建议构建和 优化按预编程顺序链接在一起的一系列模块。这项拟议的研究是由 三个具体目标：1)通过10个模块的长级联延长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信号的历史，以及3)控制 细胞记录的棘轮进程，使得细胞事件可以通过细胞分裂来识别 号码追踪器。该提案的完成将为生物学家提供大量的新能力 质疑生物系统的功能。

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.