Developing mutable barcodes for high-resolution single-cell lineage tracing

项目总结 正如基因和蛋白质表达是识别细胞的共同特征一样，谱系也是一个重要方面 细胞的身份。在过去，谱系追踪一直被用来确定哪些细胞来自特定的细胞类型， 由特定细胞类型基因的表达来定义的；然而，单细胞基因组学的建立 技术带来了单细胞分辨率的血统追踪。血统追踪的新技术可以追踪 单细胞的后代，与它们最初的基因表达无关。莫里斯实验室已经开发出一种单细胞 血统追踪(ScLT)方法，称为细胞标记。细胞标记展示了scLT方法的能力 根据谱系识别细胞的相似性，并提供对细胞重新编程的机械性见解。蜂窝标签 然而，其他基于病毒的scLT技术仍然存在局限性，因为它们需要多个 转导，以提高谱系分辨率，并可能无法捕获生物相关的分叉事件 到离散时间点的细胞标记。这些技术在某些细胞中也会受到转基因沉默的影响。 模型，如IPSC衍生的有机化合物，使其在许多开发模型中使用无效 和疾病。为了克服这些限制，有必要开发新的SCLT工具，这些工具可以在没有 重复操作细胞，并用于IPSC分化和重新编程系统，而不会沉默 阻碍了血统信息的读出。在这里，我建议使用CRISPR-Cas12a引导的胞苷 脱氨酶作为一种通过靶向胞苷连续记录可遗传谱系数据的方法 胸腺嘧啶编辑。我已经开发并验证了一种新的CRISPR-Cas12a导向胞苷的能力 脱氨酶在体外积累靶向合成DNA区域的碱基编辑并恢复这些 单细胞RNA测序合成序列(scRNA-seq)。这两个结果是一个很有希望的证据- 概念-可以使用这些累积的单一基本编辑来执行sclt。在此，我建议(1)增加 细胞标记的分辨率以捕获分叉事件，使用这种新型的DNA编辑器不断编辑单个 基于目标编辑区域(TER)，不需要多个换能器，以及(2)集成 这个碱基编辑系统被整合到IPSC品系内的一个安全港基因座上，以屏蔽转基因成分 来自沉默的技术，验证这种方法在肾脏器官分化中的作用。我建议的发展项目 细胞标记技术增加了发现的潜力，因为它们可以广泛应用于模型 系统要么不能接受多种操作，要么容易发生转基因沉默。通过制作 这些系统的所有质粒、细胞系、协议和分析工具都是公开可用的，我的目标是提供一种 多个细胞生物学领域的宝贵资源。这些资源将为以下方面提供实验工具包 任何从事体外发育、重新编程和疾病模型工作的人都可以询问单细胞 高分辨率的血统。

PROJECT SUMMARY Just as gene and protein expression are common characteristics to identify a cell, lineage is an important aspect of cell identity. In the past, lineage tracing has been used to determine what cells arise from a specific cell type, as defined by the expression of a cell-type-specific gene; however, the establishment of single-cell genomics techniques has ushered in lineage tracing at single-cell resolution. New technologies for lineage tracing can track the progeny of single cells, regardless of their initial gene expression. The Morris lab has developed a single-cell lineage tracing (scLT) method, called CellTagging. CellTagging demonstrated the ability of scLT approaches to identify similarities in cells based on lineage and offer mechanistic insights to cell reprogramming. CellTagging and other virus-based scLT technologies still present limitations, though, in that they require multiple transductions to increase lineage resolution, and may fail to capture biologically relevant bifurcation events due to cell labeling at discrete time points. These technologies are also subject to transgene silencing in certain cell models, such as iPSC-derived organoids, rendering them ineffective for use in many models of development and disease. To overcome these limitations, it is necessary to develop new scLT tools that can be applied without repeated manipulation of cells and be used in iPSC differentiation and reprogramming systems without silencing hindering the readout of lineage information. Here, I propose to utilize a CRISPR-Cas12a-guided cytidine deaminase as a method to continuously record heritable lineage data through targeted cytidine to thymine editing. I have developed and validated the ability of a novel CRISPR-Cas12a-guided cytidine deaminase to accrue base edits on a targeted synthetic DNA region over time in vitro and recovered these synthetic sequences via single-cell RNA-sequencing (scRNA-seq). These two outcomes are a promising proof- of-concept that scLT can be performed with these accrued single base edits. Here, I propose to (1) increase the resolution of CellTagging to capture bifurcation events, using this novel DNA editor to constantly edit single bases in a targeted editing region (TER) and dispense with the need for multiple transductions, and (2) integrate this base editor system into a safe harbor locus within an iPSC line to shield the transgenic components of the technology from silencing, validating this approach in kidney organoid differentiation. My proposed developments of the CellTagging technology increase the potential for discovery because they can be broadly applied to model systems that are either not amenable to multiple manipulations or are prone to transgene silencing. By making all plasmids, cell lines, protocols, and analysis tools for these systems publicly available, I aim to provide a valuable resource across several areas of cell biology. These resources will provide an experimental toolkit for anyone working with in vitro developmental, reprogramming, and disease models to interrogate single-cell lineage at high resolution.