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

基于 CRISPR/Cas9 的谱系追踪用于癌细胞和干细胞的命运图谱

基本信息

批准号：
9262180
负责人：
Zhe Li
金额：
$ 20.74万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-15 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9262180
关键词：
Address Adenoviruses Affect Alpha Cell Animal Model Behavior Breast Cancer cell line Breast Epithelial Cells CRISPR/Cas technology Cell Culture Techniques Cell Lineage Cells Coupled Environment Epigenetic Process Genetic Genomic Segment Goals Guide RNA Habitats Heterogeneity Human Human Genome In Vitro Injection of therapeutic agent Intestinal Cancer Knowledge Label MDA MB 231 Malignant Neoplasms Malignant neoplasm of brain Mammary gland Maps Methods Mus Mutation One-Step dentin bonding system Physiologic pulse Physiological Population Preclinical Drug Evaluation Resistance Response Elements Skin Cancer Solid Neoplasm Somatic Cell Specificity Stem cells System Testing Tissues Translating Transplantation Tumor Tissue Writing Xenograft Model Xenograft procedure anticancer research base cancer cell cancer genomics cancer stem cell cancer therapy cell behavior cell type chemotherapy drug testing endonuclease genetic approach genome editing in vivo insertion/deletion mutation interest malignant breast neoplasm mouse genome mouse model next generation sequencing novel novel strategies prevent promoter public health relevance recombinase-mediated cassette exchange stem stem cell population success therapy resistant tumor tumor progression

项目摘要

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.

描述（由适用提供）：尽管细胞培养通常用于在体外研究癌细胞和干细胞，但在其本机环境下，这些细胞的行为与体内的培养皿与体内的行为可能完全不同。谱系追踪是一种在动物模型中经常使用的遗传方法，用于确定癌细胞或组织干细胞在生理环境下的行为，但其对人类细胞的使用受到限制。该项目的目的是开发小说谱系追踪系统能够启用人（癌症）细胞的一步遗传标记（例如，在异种移植小鼠模型中），能够标记可以在体内遵循的细胞的预定细胞子集标记其克隆行为，以免在其体内引入突变（由于遗传标记），从而影响了相同的型号，从而影响了collonal行为，并且可以构图collos/collation collation collation collation collation collanal carlonal collation and collanal collation and collos collation and coly collation and collotal collation collos and collanal conter and collonal conter and vi for f ivo。简单的组织/肿瘤。由于CRISPR/CAS9基因组编辑系统可以有效地“写入”人类或小鼠基因组预先指定的区域中的插入或缺失（indel）突变，因此假设这种随机的indeel突变本身可以用作稳定的条形线条跟踪目标的条形码。 CAS9表达可以以细胞类型的特异性控制以实现细胞标记的特异性。我们组已验证组织干细胞脉冲/追逐谱系研究的腺病毒系统可用于在体内提供CRISPR/CAS9系统。提出了两个具体的目的来解决这一假设：1）通过多重谱系追踪在同一组织中的多个干细胞种群对体内特定细胞谱系的映射贡献：自从引入indel突变（即“ Bar codes”（即“ Barcodes”）之后基于CRISPR/CAS9的系统可能是其多路复用谱系跟踪的能力。为了测试这一想法，将在体内的小鼠乳腺上皮细胞中引入indel突变（因此引入的突变是中性的），以确定乳腺腔谱系是由单肌管腔干细胞还是双能干细胞或两者在物理设置下维持的乳腺腔谱系。 2）在异种移植模型中绘制人类乳腺癌干细胞（CSC）的体内命运：在这里，基于CRISPR/CAS9的谱系追踪在人类细胞中的可行性将通过向人类基因组中的AAVS1安全港基因座引入indel indel突变来确定。腺病毒将用于在人类乳腺癌细胞系M MDA-MB-231中，将CRISPR/CAS9传递给CSC种群（根据由SOX2/OCT4-RESPONSE元件的重复序列组成的合成启动子定义）。 CSC的克隆动力学与大部分由CAS9进行的癌细胞进行了比较，将在具有或不进行化学疗法的异种移植模型中进行比较。如果成功，这种新颖的谱系追踪方法将是一种变化，可以增强我们对癌细胞如何从其体内原始细胞演变的理解以及它们如何对治疗具有抵抗力。