A cell-cycle induced genetic recorder for simultaneous recovery of cell divisions and lineage

细胞周期诱导的遗传记录仪，用于同时恢复细胞分裂和谱系

基本信息

批准号：
10358198
负责人：
ANGELA M. BELCHER
金额：
$ 29.72万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10358198
关键词：
Address Animals Bar Codes Cancer Biology Cancer Model Cell Cycle Cell Line Cell Lineage Cell division Cells Clonal Evolution Clonality Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA Development Developmental Biology Devices Engineering Evolution Generations Genes Genetic Genome Genomics Heritability Human Human Cloning Individual Intention Intervention Light Liver Longevity Longitudinal Studies Malignant Neoplasms Maps Mediating Methods Modeling Molecular Mus Organism Organoids Pattern Physiological Population Population Dynamics Process Records Recovery Relapse Resistance Source System Technology Time Translational Research Ubiquitination base cancer stem cell design effective therapy genomic locus improved in vivo induced pluripotent stem cell live cell imaging liver cancer model loss of function new therapeutic target prime editing programs reconstruction response self-renewal single-cell RNA sequencing synthetic biology therapy resistant transcriptomics tumor tumor growth tumor initiation tumor progression

项目摘要

Project Summary Revealing the lineage relations among cells has the potential to illustrate evolutionary patterns in tumor initiation and resistance to therapies and shed light on the processes which give rise to developmentally complex organisms. However, studying these relationships in cancer biology and developmental biology is currently limited by the difficulty of cell lineage tracing and reconstruction in-vivo. Complete reconstructions of cell lineages require non-invasive live cell imaging, which is restricted to relatively small numbers of cells and impractical for physiologically-relevant studies. Although recent systems for CRISPR-mediated genome barcoding have emerged as a promising alternative, they suffer limitations in the diversity and generation rate of lineage- identifying information which render them unsuited to studies of clonal evolution and lineage development, especially in cancer. Moreover, these methods fail to explicitly capture cell divisions, a basic parameter of somatic evolution which could impact our understanding of a cancer's response to intervention. To improve recovery of population dynamics and cell lineage information from cancer models, we propose a cell- cycle inducible genetic construct that will record, in a single locus, the sequence of divisions and lineage of each cell. Our approach uses cell-cycle regulated ubiquitination to control the activity of a donor-free CRISPR-based genome recording device which barcodes cells upon each division. In this study, we will evaluate the ability of our device to track lineage information and cell division dynamics in clonal human and mouse cell populations. To demonstrate its investigative potential in developmental studies, we will combine our cell-division barcoding strategy with single-cell RNA sequencing to identify healthy and abnormal differentiation in cancer- and hiPSC- derived organoids. If successful, our improvements to recovery of cell division, clonality, and lineage will improve our understanding of development, explain evolutionary sources of resistance in cancer, and guide practitioners toward more effective treatments in translational research.

项目概要揭示细胞之间的谱系关系有可能阐明肿瘤发生的进化模式和对治疗的抵抗力，并揭示了导致发育复杂的过程有机体。然而，目前正在研究癌症生物学和发育生物学中的这些关系受限于体内细胞谱系追踪和重建的难度。细胞谱系的完整重建需要非侵入性活细胞成像，这仅限于相对较少数量的细胞，并且对于生理相关的研究。尽管最近 CRISPR 介导的基因组条形码系统已经作为一种有前途的替代方案出现，它们在谱系的多样性和生成率方面受到限制识别使它们不适合克隆进化和谱系发展研究的信息，尤其是在癌症方面。此外，这些方法无法明确捕获细胞分裂，这是细胞分裂的基本参数。体细胞进化可能会影响我们对癌症对干预反应的理解。为了改善癌症模型中群体动态和细胞谱系信息的恢复，我们提出了一种细胞- 循环诱导遗传构建体将在单个基因座中记录每个细胞的分裂序列和谱系细胞。我们的方法使用细胞周期调节的泛素化来控制基于无供体 CRISPR 的活性基因组记录装置，在每次分裂时对细胞进行条形码标记。在本研究中，我们将评估以下人员的能力：我们的设备用于跟踪克隆人类和小鼠细胞群的谱系信息和细胞分裂动态。为了展示其在发育研究中的研究潜力，我们将结合我们的细胞分裂条形码利用单细胞 RNA 测序识别癌症和 hiPSC 中健康和异常分化的策略衍生的类器官。如果成功，我们对细胞分裂、克隆性和谱系恢复的改进将得到改善我们对发展的理解，解释癌症耐药性的进化来源，并指导从业者在转化研究中寻求更有效的治疗方法。