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

 描述（由申请人提供）：尽管细胞培养通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与在其天然环境下的体内行为可能完全不同。谱系追踪是一种遗传学方法，常用于动物模型中，以确定生理环境下癌细胞或组织干细胞的行为，但其用于人类细胞是有限的。这个项目的目的是开发一部小说 谱系追踪系统能够一步遗传标记人（癌）细胞（例如，在异种移植小鼠模型中），以能够标记可在体内跟踪其克隆行为的预定义细胞亚群，不引入可能影响克隆行为的突变（由于遗传标记），并具有同时绘制相同组织/肿瘤中多种干细胞/癌细胞群体的体内命运的能力。由于CRISPR/Cas9基因组编辑系统可以在人或小鼠基因组的预先指定的区域中有效地“写入”插入或缺失（indel）突变，因此假设这种随机indel突变本身可以用作用于谱系追踪目的的稳定条形码。可以以细胞类型特异性方式控制Cas9表达以实现细胞标记特异性。我们的研究小组已经验证了一种腺病毒系统，用于组织干细胞的脉冲/追踪谱系追踪研究，该系统可用于体内递送CRISPR/Cas9系统。提出了两个具体的目的来解决这个假设：1）通过多重谱系追踪在体内映射相同组织内的多个干细胞群体对特定细胞谱系的贡献：由于在引入插入缺失突变（即，尽管基于CRISPR/Cas9的系统可以通过CRISPR/Cas9对不同的细胞类型或不同的基因组区域进行多重谱系追踪（“条形码”），但是这样的条形码可以通过下一代测序以平行方式解码，基于CRISPR/Cas9的系统的重要特征可以是其多重谱系追踪的能力。为了测试这个想法，将插入缺失突变引入到体内小鼠乳腺上皮细胞中的安全港Rosa 26基因座（因此引入的突变是中性的），以确定在生理环境下，乳腺腔谱系是由单能腔干细胞或双能基底干细胞或两者维持。2)在异种移植模型中绘制人乳腺癌干细胞（CSC）的体内命运：在这里，将通过向人类基因组中的AAVS 1安全港基因座引入indel突变来确定在人类细胞中基于CRISPR/Cas9的谱系追踪的可行性。腺病毒将用于将CRISPR/Cas9递送至人乳腺癌细胞系MDA-MB-231中的CSC群体（基于由S 0X 2/0 CT 4-反应元件的重复组成的合成启动子定义），用于条形码化。将在有或没有化疗的异种移植模型中或在连续移植后比较CSC相对于由Cas9条形码化的癌细胞的体积的克隆动力学。如果成功的话，这种新的谱系追踪方法将是变革性的，可以提高我们对癌细胞如何从体内起源细胞进化以及它们如何对治疗产生抗性的理解。

 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.