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(造血干细胞的分化)与心血管疾病的进展 疾病(心血管疾病)。为了缩小这一知识差距，该计划项目将把领域领先的 研究人员调查目前未知的因素，介导CVD过程后的造血 包括心肌梗死、动脉粥样硬化和中风，同时也对这些疾病的解剖贡献 影响CVD进展的因素。因此，有一个很好的机会来利用 像基因组编辑这样的平台技术可以在体外设计HSCs，并在CVD的小鼠模型中帮助 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 序列变体的功能意义。基因组编辑使永久可遗传的改变成为可能 通过在特定序列处诱导DNA中的定向双链断裂(DSB)来实现的遗传序列。一个 被称为表观基因组编辑的互补方法利用核酸酶的DNA靶向能力 通过招募异源效应域来瞬时改变基因表达的平台 将转录状态调节到基因组的调节区，如启动子或增强子，而不需要 对DSB的需求。因此，我们基因组工程核心(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、安全和 高效编辑HSCs以阐明造血和CVD之间的联系。的主要目标 核心2包括通过优化实验方法支持该提案的项目，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，以 描述造血干细胞的基因、过程和分化。具体地说，核心2将通过以下方式支持项目1-4 通过开发新的表观基因组，在小鼠HSCs中实现高效和特定的基因组编辑策略 编辑试剂用于人类干细胞，并通过实施和开发基于编辑的血统追踪方法。 该核心所追求的鼠标HSC编辑的成功优化和开发将加速 项目1至4能够检查造血和心血管疾病之间的关系，以及更广泛地说， 这些进展将广泛应用于心血管疾病的研究和治疗干预。 造血障碍，以及造血干细胞的体外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.