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

摘要 克隆突起广泛存在于正常人体组织中。克隆人窝藏体细胞 已知癌症和其他驱动基因的突变，并显示出正向选择的证据。然而，如何 这些驱动程序突变改变了细胞的细胞状态，使克隆人能够与野生型竞争对手竞争 人们对此仍然知之甚少。到目前为止，绘制正常组织中克隆突起的努力主要是 仅限于基因分型。这是因为克隆通常影响样本中的少数细胞，而不是 区分细胞表面标记或形态特征的。 为了应对这一挑战，我们开发了一系列多组体单细胞技术，能够 获取多层信息(例如，基因类型、转录本、甲基组、蛋白质表达) 同样的单细胞。此外，我们还解决了单细胞scrna-seq基因分型的特殊挑战。 通过发展转录本的基因分型，实现细胞的高通量。重要的是，这项技术将 突变细胞和野生型细胞的混合从限制到优势，使直接比较 同一个体内的突变(“赢家”)和野生型(“输家”)细胞。 利用我们在单细胞技术开发方面的经验，我们的目标是扩大多组学 单细胞GET(靶点基因分型)工具包，允许询问体细胞突变是如何导致 克隆生长优势。首先，我们将在此背景下发展和加强我们的目标单细胞基因分型 染色质可及性(GOT-CHA)。这项技术关键是直接从DNA中进行基因分型， 避免了对突变基因表达的有限依赖。因此，它可以应用于提取的细胞核， 对SMAHTT倡议至关重要。我们将在GOT-CHA的基础上使用纳米体拴系转座酶联合 单核的体细胞突变和组蛋白修饰(GOT-EPIM)。捕获转录的 结合体细胞突变基因分型和染色质可及性，我们将进一步使用 信使核糖核酸转座：GOT-CHA-RNA中的cDNA杂交体。最后，我们将利用使用 用寡核苷酸标记的抗体捕获突变的基因座、染色质和核内蛋白，如 转录因子(GOT-CHA-Pro)。在目标2中，我们将与基因组表征中心合作 将这些技术应用于原生人类样本，以确定正常组织中的克隆突变是如何改变的 染色质、组蛋白修饰、转录本和蛋白质丰度谱以产生克隆副产物。 我们的首要目标是在单细胞水平上对野生型和突变型进行多组比较。 细胞，以全面识别克隆生长中适应性优势的基础。建议数 全面的GET工具包将使整个单细胞基因型别与转录， 蛋白质和表观遗传学，在克隆嵌合体作为癌症先兆的研究中具有重要意义，如 以及其他人类健康结果。

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.