Temporal recording of mammalian development and precancer [WES]. Temporal recording of mammalian development and precancer [WES]

Insights into many biological phenomena requires knowing the temporal order of cellular events, which is traditionally achieved through continuous direct observations [1, 2]. An alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using Native sgRNA Capture and sequencing (NSC-seq), a newly devised and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo while incorporating cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development, unconventional developmental relationships between cell types, and new epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas coupled to multi-omic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses to explore the origins and timing of development and tumorigenesis in mammalian systems. Overall design: DNA was extracted from resected tumors and normal epithelial tissues using DNA Extraction Kits (Qiagen). Standard whole exome sequencing (WES) library was prepared using mouse exome panel (Twist Bioscience) and sequenced on S4 flow cells on NovaSeq6000 (PE150) to the targeted coverage (50X). WES reads were aligned to the mouse reference genome (mm10) using BWA and indexed by Sambamba. Somatic variants were called using GATK Mutect2. Variants were annotated using ANNOVAR and germline variants were filtered using paired normal samples. Variants were further analyzed and visualized using Maftools.

对诸多生物学现象的深入解析，有赖于明确细胞事件的时间顺序——传统上该信息需通过持续直接观测获取[1,2]。另一种解决方案则借助不可逆的遗传改变（如自然发生的突变）构建不可磨灭的标记物，以此实现回溯性的时间排序[3-8]。本研究依托全新开发并验证的多用途单细胞成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）平台——原生sgRNA捕获测序（Native sgRNA Capture and sequencing, NSC-seq），开发了一种分子时钟方法，可在体内记录细胞事件的发生时序与克隆性，同时整合细胞状态与谱系信息。借助该方法，我们精准解析了小鼠胚胎发育过程中组织特异性细胞扩增的精确时序，揭示了细胞类型间非常规的发育关联，以及基于独特遗传历史的新型上皮祖细胞状态。我们对小鼠腺瘤开展NSC-seq分析，并结合人类癌前病变的多组学与单细胞谱分析，同时对418例人类息肉开展克隆分析，结果证实15%~30%的结直肠癌前病变存在多祖先起源，揭示其源自多个正常创始细胞。本研究提出了一个多模态研究框架，为体内记录奠定了基础——该框架将合成或天然的不可磨灭遗传改变与单细胞分析相结合，用以探索哺乳动物系统中发育与肿瘤发生的起源与时序。整体实验设计：使用DNA提取试剂盒（Qiagen）从切除的肿瘤与正常上皮组织中提取基因组DNA。采用小鼠外显子组捕获面板（Twist Bioscience）构建标准全外显子测序（whole exome sequencing, WES）文库，并在NovaSeq6000测序平台的S4流动槽上以PE150模式进行测序，以达到靶向覆盖度（50X）。使用BWA将WES读段比对至小鼠参考基因组（mm10），并通过Sambamba进行索引。采用GATK Mutect2进行体细胞变异检出。通过ANNOVAR对变异进行注释，并利用配对正常样本过滤种系变异。后续借助Maftools对变异进行进一步分析与可视化。