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

开发用于高分辨率单细胞谱系追踪的可变条形码

• 批准号: 10536930
• 负责人: Sadie M VanHorn
• 金额: $ 3.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10536930
• 关键词: Address; Animals; Area; Bar Codes; Biological Models; Biological Process; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cell Lineage; Cell Transplantation; Cell model; Cells; Cellular biology; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Cytidine; Cytidine Deaminase; DNA; Data; Development; Disease; Disease model; Event; Gene Expression; Genes; Genome; Genomics; Heritability; In Vitro; Individual; Intervention; Kidney; Label; Lentivirus; Methods; Modeling; Mutate; Mutation; Organoids; Outcome; Plasmids; Population; Protocols documentation; Resolution; Resources; System; Techniques; Technology; Thymine; Time; Transcript; Transgenes; Transgenic Organisms; Validation; Viral; Virus; Work; Writing; base; base editing; base editor; cell type; clinically relevant; discrete time; induced pluripotent stem cell; insight; invention; model development; new technology; novel; protein expression; reconstruction; single-cell RNA sequencing; synthetic construct; tool

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.

项目摘要 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，谱系追踪已被用于确定哪些细胞来自特定的细胞类型， 如细胞类型特异性基因的表达所定义的;然而，单细胞基因组学的建立 技术已经引入了单细胞分辨率的谱系追踪。血统追踪的新技术可以追踪 单细胞的后代，不管它们最初的基因表达如何。莫里斯实验室已经开发出一种单细胞 谱系追踪（scLT）方法，称为CellTagging。CellTagging证明了scLT方法能够 基于谱系识别细胞中的相似性，并为细胞重编程提供机制见解。单元格标记 和其他基于病毒的scLT技术仍然存在局限性，因为它们需要多个 转导以增加谱系分辨率，并且可能无法捕获生物学相关的分叉事件， 到离散时间点的细胞标记。这些技术在某些细胞中也受到转基因沉默的影响。 模型，如iPSC衍生的类器官，使它们在许多发育模型中无效 和疾病为了克服这些限制，有必要开发新的scLT工具， 重复操作细胞并用于iPSC分化和重编程系统而不沉默 阻碍了谱系信息的读出。在这里，我建议利用CRISPR-Cas 12 a指导的胞苷 脱氨酶作为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型CRISPR-Cas 12 a引导的胞苷 脱氨酶在体外随着时间的推移在靶向合成DNA区域上累积碱基编辑，并回收这些 通过单细胞RNA测序（scRNA-seq）合成序列。这两个结果是一个有希望的证明- 从概念上讲，scLT可以用这些累积的单碱基编辑来执行。在此，我建议（1）增加 解决CellTagging捕获分叉事件，使用这种新颖的DNA编辑器不断编辑单个 靶向编辑区（TER）中的碱基，并免除多次转导的需要，和（2）整合 将该碱基编辑系统插入iPSC系内的安全港基因座中，以屏蔽iPSC的转基因组分。 技术沉默，验证这种方法在肾类器官分化。我提议的发展 CellTagging技术增加了发现的潜力，因为它们可以广泛应用于模型 这些系统要么不适合于多次操作，要么倾向于转基因沉默。通过使 所有质粒，细胞系，协议和分析工具，这些系统公开，我的目的是提供一个 细胞生物学多个领域的宝贵资源。这些资源将提供一个实验工具包， 任何从事体外发育、重编程和疾病模型研究的人， 高分辨率的血统。