Single-Cell Multi-omics to Link Clonal Mosaicism (CM) Genotypes with Chromatin, Epigenomic, Transcriptomic and Protein Phenotypes

单细胞多组学将克隆嵌合 (CM) 基因型与染色质、表观基因组、转录组和蛋白质表型联系起来

• 批准号: 10662879
• 负责人: Dan Landau
• 金额: $ 42.82万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662879
• 关键词: ATAC-seq; Address; Admixture; Affect; Antibodies; Bar Codes; Biological Assay; Biology; Cell Nucleus; Cell physiology; Cell surface; Cells; Chromatin; Clonal Expansion; Collaborations; Complementary DNA; Complex; Coupled; DNA; DNA Sequence Alteration; Data; Dependence; Ensure; Epigenetic Process; Freezing; Gene Expression; Genes; Genetic; Genetic Heterogeneity; Genetic Transcription; Genomic DNA; Genomics; Genotype; Goals; Growth; Health; Hematopoiesis; Histones; Human; Human body; Hybrids; In Situ; Individual; Knowledge; Lead; Libraries; Link; Malignant Neoplasms; Maps; Measurement; Messenger RNA; Methods; Minority; Modification; Morphology; Mosaicism; Mutate; Mutation; Nature; Normal Range; Normal tissue morphology; Oligonucleotides; Outcome; Phase; Phenotype; Population; Proteins; RNA; Recurrence; Resolution; Reverse Transcription; Sampling; Signal Transduction; Somatic Mutation; Technology; Tissues; Transposase; Work; cancer diagnosis; cell behavior; cell type; clinical diagnosis; differential expression; driver mutation; epigenome; epigenomic profiling; epigenomics; experience; fitness; genomic locus; histone modification; human tissue; mRNA Expression; methylome; multiple omics; mutant; nanobodies; novel; programs; protein expression; single cell sequencing; single cell technology; single-cell RNA sequencing; technology development; tool; transcription factor; transcriptome; transcriptomics

SUMMARY Clonal outgrowths are observed across a wide range of normal human tissues. Clones harbor somatic mutations in known cancer and other driver genes, and show evidence of positive selection. Nevertheless, how these driver mutations alter the cellular states of cells to allow clones to outcompete wildtype counterparts remains poorly understood. To date, efforts to chart clonal outgrowths in normal tissues have been largely limited to genotyping. This is due to the fact that clones often affect a minority of cells in a sample, without distinguishing cell surface markers or morphological features. To address this challenge, we developed an array of multi-omic single-cell technologies that are capable of capturing multiple layers of information (e.g., genotypes, transcriptomes, methylomes, protein expression) from the same single cells. Moreover, we addressed the specific challenge of genotyping in scRNA-seq in single cells at high throughput by developing genotyping of transcriptomes. Importantly, this technology turns the admixture of mutant and wildtype cell from a limitation to an advantage, enabling the direct comparison of mutant (“winner”) and wildtype (“loser”) cells within the same individual. Capitalizing on our experience with single-cell technology development, we aim to extend the multi-omics single-cell GoT (Genotyping of Targeted loci) toolkit to allow to interrogate how somatic mutations lead to clonal growth advantage. First, we will develop and enhance our targeted single-cell genotyping in the context of chromatin accessibility (GoT-ChA). This technology critically performs genotyping from DNA directly, obviating limiting dependencies on mutated loci gene expression. Thus, it can be applied to extracted nuclei, critical for the SMaHT initiative. We will build on GoT-ChA using nanobody tethered transposases to jointly profile somatic mutations and histone modifications in single nuclei (GoT-EpiM). To capture transcriptional changes together with somatic mutation genotyping and chromatin accessibility, we will further use transposition of mRNA:cDNA hybrid in GoT-ChA-RNA. Finally, we will leverage recent advances that use antibodies tagged with oligonucleotides to capture mutated loci, chromatin and intra-nuclear proteins such as transcription factors (GoT-ChA-Pro). In aim 2, we will collaborate with genomic characterization centers to apply these technologies to primary human samples to define how clonal mutations in normal tissues alter chromatin, histone modifications, transcriptomes and protein abundance profiles to yield clonal outgrowth. Our overarching goal is to invoke multi-omic comparisons at the single-cell level between wildtype and mutant cells to comprehensively identify the underpinnings of fitness advantage in clonal outgrowth. The proposed comprehensive GoT toolkit will enable to link, at high throughout single-cell genotypes with transcriptional, protein, and epigenetic, with important implication in the study of clonal mosaicism as a harbinger of cancer, as well as other human health outcomes.

概括 在广泛的正常人体组织中观察到克隆产物。克隆藏有躯体 已知癌症和其他驱动器基因的突变，并显示出阳性选择的证据。然而，怎么样 这些驱动器突变改变了细胞的细胞状态，使克隆胜过野外型 仍然很了解。迄今为止，绘制正常组织中克隆出生生长的努力在很大程度上已经很大 限于基因分型。这是由于以下事实：克隆通常会影响样品中的少数细胞，而没有 区分细胞表面标记或形态特征。 为了应对这一挑战，我们开发了一系列能够 从中捕获多层信息（例如，基因型，转录组，甲基组，蛋白质表达） 相同的单个单元格。此外，我们解决了单个SCRNA-SEQ中基因分型的具体挑战 通过发展转录组的基因分型，处于高通量的细胞。重要的是，这项技术使 突变体和野生型细胞从限制到优势的混合，使得直接比较 突变体（“赢家”）和野生型（“失败者”）细胞内。 利用我们在单细胞技术开发方面的经验，我们旨在扩展多摩ic 单细胞获得（靶向基因座的基因分型）工具包，以询问体细胞突变如何导致 克隆生长优势。首先，我们将在上下文中开发和增强我们的目标单细胞基因分型 染色质可及性（GOT-CHA）。这项技术严重地从DNA直接执行基因分型， 消除对突变基因座基因表达的限制依赖性。那就可以应用于提取的核， 对SMAHT倡议至关重要。我们将使用纳米型束缚转座酶建立在got-cha上 单核（GOT-EPIM）中的体细胞突变和组蛋白修饰。捕获转录 随着体细胞突变基因分型和染色质的可及性的变化，我们将进一步使用 mRNA的转座：got-cha-rna中的cDNA杂交。最后，我们将利用使用的最新进展 用寡核苷酸标记的抗体以捕获突变的基因座，染色质和核内蛋白（例如 转录因子（got-cha-pro）。在AIM 2中，我们将与基因组表征中心合作 将这些技术应用于主要人类样品，以定义正常组织中的克隆突变如何改变 染色质，组蛋白修饰，转录组和蛋白质抽象谱，以产生克隆生长。 我们的总体目标是在WildType和突变体之间在单细胞水平上调用多OMIC比较 细胞全面识别克隆出生中健身优势的基础。提议 全面的获得工具包将使整个单细胞基因型具有转录， 蛋白质和表观遗传学，在克隆镶嵌作为癌症预兆的研究中具有重要意义 以及其他人类健康结果。