CRISPR/Cas9-based lineage tracing for fate mapping of cancer cells and stem cells

 描述(申请人提供)：虽然细胞培养经常用于体外研究癌细胞和干细胞，但这些细胞在培养皿中的行为可能与在其自然环境下的体内行为截然不同。谱系追踪是一种经常用于动物模型的遗传学方法，用于确定癌细胞或组织干细胞在生理环境下的行为，但它在人类细胞中的应用有限。这个项目的目的是开发一种新的 血统追踪系统能够对人类(癌症)细胞(例如在异种移植小鼠模型中)进行一步遗传标记，能够标记体内可跟踪其克隆行为的预定义细胞子集，不会引入可能影响克隆行为的突变(由于基因标记)，并有能力同时绘制同一组织/肿瘤中多个干细胞/癌细胞群体的体内命运图。由于CRISPR/Cas9基因组编辑系统可以有效地将插入或缺失(Indel)突变写入人类或小鼠基因组的预先指定区域，因此可以假设这种随机Indel突变本身可以作为稳定的条形码用于谱系追踪目的。Cas9的表达可以以细胞类型特异性的方式进行控制，以实现细胞标记的特异性。我们的团队已经验证了一种腺病毒系统，用于组织干细胞的脉冲/追赶谱系跟踪研究，该系统可以用于体内传递CRISPR/Cas9系统。提出了两个具体的目标来解决这一假设：1)通过多重谱系追踪，将同一组织内的多个干细胞群体在体内对特定细胞谱系的贡献映射到特定的细胞谱系：由于当CRISPR/Cas9将插入突变(即“条形码”)引入不同的细胞类型或不同的基因组区域时，这种条形码可以通过并行的下一代测序方式解码，因此基于CRISPR/Cas9的系统的一个重要特征可能是其多重谱系追踪的能力。为了验证这一想法，将在体内将插入突变引入小鼠乳腺上皮细胞的安全港rosa26基因座(因此引入的突变是中性的)，以确定在生理环境下，乳房管腔血统是由单能的管腔干细胞还是双能的基础干细胞维持的，或者两者兼而有之。2)在异种移植模型中定位人乳腺癌干细胞(CSCs)的体内命运：在这里，基于CRISPR/Cas9的人类细胞谱系追踪的可行性将通过在人类基因组中引入AAVS1安全港基因来确定。腺病毒将被用来将CRISPR/Cas9转移到人乳腺癌细胞系MDA-MB-231中的CSC群体(基于由SOX2/OCT4反应元件的重复组成的合成启动子)进行条形码。在有或没有化疗或连续移植的异种移植模型中，将比较CSCs与Cas9标记的大部分癌细胞的克隆动力学。如果成功，这种新的谱系追踪方法将有助于增强我们对癌细胞如何在体内从起源细胞进化以及它们如何对治疗产生抵抗力的理解。

 DESCRIPTION (provided by applicant): Although cell culture is often used to study cancer cells and stem cells in vitro, behaviors of these cells may be quite different in culture dishes versus in vivo under their native environment. Lineage tracing is a genetic approach often used in animal models to determine behaviors of cancer cells or tissue stem cells under physiological settings, but its use for human cells is limited. The purpose of this project is to develop a novel lineage-tracing system to enable one-step genetic marking of human (cancer) cells (e.g., in xenograft mouse model), to enable labeling of a pre-defined subset of cells that can be followed in vivo for their clonal behavior, to not introduce mutations (due to genetic marking) that may affect clonal behavior, and to have the capacity to map in vivo fates of multiple stem cell/cancer cell populations in the same tissue/tumor simultaneously. Since the CRISPR/Cas9 genome-editing system can efficiently "write" insertion or deletion (indel) mutations in pre-designated regions of the human or mouse genome, it is hypothesized that such random indel mutations themselves can serve as stable barcodes for lineage-tracing purpose. Cas9 expression can be controlled in a cell type-specific manner to achieve cell-labeling specificity. An adenoviral system that has been validated by our group for pulse/chase lineage-tracing studies of tissue stem cells can be used to deliver the CRISPR/Cas9 system in vivo. Two Specific Aims are proposed to address this hypothesis: 1) Map contributions of multiple stem cell populations within the same tissue to a specific cell lineage in vivo by multiplexed lineage tracing: Since upon introduction of indel mutations (i.e., "barcodes") by CRISPR/Cas9 to different cell types or to different genomic regions, such barcodes can be decoded by next-generation sequencing in a parallel fashion, an important feature of the CRISPR/Cas9-based system may be its capacity for multiplexed lineage tracing. To test this idea, indel mutations will be introduced to the safe-harbor Rosa26 locus (thus mutations introduced are neutral) in mouse mammary epithelial cells in vivo, to determine whether the mammary luminal lineage is maintained by unipotent luminal stem cells, or bipotent basal stem cells, or both, under the physiological setting. 2) Map the in vivo fate of human breast cancer stem cells (CSCs) in a xenograft model: here the feasibility of CRISPR/Cas9-based lineage tracing in human cells will be determined by introducing indel mutations to the AAVS1 safe harbor locus in the human genome. Adenovirus will be used to deliver CRISPR/Cas9 to a CSC population (defined based on a synthetic promoter composed of repeats of SOX2/OCT4-response element) in the human breast cancer cell line, MDA-MB-231, for barcoding. Clonal dynamics of CSCs versus the bulk of cancer cells barcoded by Cas9 will be compared in a xenograft model with or without chemotherapy, or upon serial transplantation. If success, this novel lineage-tracing approach would be transformative for enhancing our understanding of how cancer cells evolve from their cells of origin in vivo and how they become resistant to therapy.