Core 2: GENETIC ENGINEERING CORE

摘要 本计划项目的核心目标是定义以下各项之间的关系和相互联系： 造血（造血干细胞或HSC的分化）和心血管疾病的进展 疾病（CVD）。为了缩小这一知识差距，该计划项目将汇集外地领先的 研究人员调查目前未知的因素，介导血细胞生成以下CVD过程 包括心肌梗死、动脉粥样硬化和中风，同时也是这些疾病的解剖学贡献。 CVD进展的因素。因此，有很大的机会利用 平台技术，如基因组编辑，在体外和CVD小鼠模型中改造HSC， 解决这些悬而未决的问题。基因组编辑技术的进步使研究人员能够 精确地改变DNA序列和基因表达，允许快速生成生物模型来测试 序列变异的功能意义。基因组编辑使永久性遗传改变成为可能。 通过在特定序列处诱导DNA中的靶向双链断裂（DSB）来改变基因序列。一 一种称为表观基因组编辑的互补方法利用了核酸酶的DNA靶向能力 通过募集能够瞬时改变基因表达的异源效应结构域的平台， 将转录状态调节到基因组的调节区域，如启动子或增强子，而不 对DSB的需求。因此，我们的基因组工程核心（核心2）的长期目标是同时 开发和优化基因组和表观基因组编辑技术，使快速，安全， HSC的有效编辑以阐明造血与CVD之间的联系。大目标 核心2包括通过优化实验方法支持本提案的项目，并提供 基因组和表观基因组编辑试剂和项目1至4的关键实验的专业知识。核心2将 还开发新颖和创新的基因组编辑，表观基因组编辑和谱系追踪平台， 表征HSC的基因、过程和分化。具体而言，核心2将支持项目1-4， 通过开发新的表观基因组，在小鼠HSC中实现有效和特异性的基因组编辑策略， 用于HSC的编辑试剂，以及通过实施和开发基于编辑的谱系追踪方法。 该核心所追求的小鼠HSC中编辑的成功优化和开发将加快 项目1至4的能力，以检查造血和CVD之间的关系，更广泛地说， 这些进展将广泛应用于心血管疾病的研究和治疗干预， 造血系统疾病，以及HSC的离体基因组和表观基因组编辑。

ABSTRACT The core objective of this Program Project is to define the relationships and interconnections between hematopoiesis (the differentiation of hematopoietic stem cells, or HSCs) and the progression of cardiovascular diseases (CVDs). To close this gap in knowledge, the Program Project will bring together field-leading researchers to investigate currently unknown factors that mediate hematopoiesis following CVD processes including myocardial infarction, atherosclerosis, and stroke, while also the dissecting contribution of these factors to CVD progression. Therefore, there exists a great opportunity to leverage the capabilities imparted by platform technologies like genome editing to engineer HSCs ex vivo and in mouse models of CVD to help address these outstanding questions. Advances in genome-editing technologies have enabled researchers to precisely alter DNA sequences and gene expression, allowing the rapid generation of biological models to test functional significance of sequence variants. Genome editing enables the permanent heritable alteration of genetic sequence by inducing targeted double-strand breaks (DSBs) in DNA at specified sequences. A complementary approach called epigenome editing leverages the DNA targeting capabilities of the nuclease platforms to transiently alter gene expression by recruiting heterologous effector domains capable of modulating transcriptional states to regulatory regions of the genome, such as promoters or enhancers, without the need for DSBs. Thus, the long-term goal of our Genome Engineering Core (Core 2) is to concurrently develop and optimize genome- and epigenome editing technologies that will enable the rapid, safe, and efficient editing of HSCs to elucidate the connection between hematopoiesis and CVD. Major objectives of Core 2 include supporting the Projects of this proposal by optimizing experimental approaches, and providing genome and epigenome editing reagents and expertise for key experiments of Projects 1 through 4. Core 2 will also develop novel and innovative genome editing, epigenome editing, and lineage tracing platforms to characterize genes, processes, and differentiation of HSCs. Specifically, Core 2 will support Projects 1-4 by enabling efficient and specific genome editing strategies in mouse HSCs, by developing novel epigenome editing reagents for use in HSCs, and by implementing and developing editing-based lineage tracing methods. Successful optimization and development of editing in mouse HSCs pursued by this Core will expedite the ability of Projects 1 through 4 to examine the relationships between hematopoiesis and CVD and more broadly, these advances will be widely applicable to the study and therapeutic intervention of cardiovascular and hematopoietic disorders, and the ex vivo genome and epigenome editing of HSCs.