Core 2: GENETIC ENGINEERING CORE

摘要 该计划项目的核心目标是定义之间的关系和互连 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，该计划项目将汇集领域领先的 研究人员将调查目前未知的介导 CVD 过程后造血作用的因素 包括心肌梗塞、动脉粥样硬化和中风，同时也包括这些疾病的解剖贡献 CVD进展的因素。因此，存在一个很好的机会来利用所赋予的能力 基因组编辑等平台技术可在体外和小鼠心血管疾病模型中设计造血干细胞，以帮助 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，从而快速生成用于测试的生物模型 序列变异的功能意义。基因组编辑可以实现永久可遗传的改变 通过在 DNA 的特定序列处诱导目标双链断裂 (DSB) 来获得基因序列。一个 称为表观基因组编辑的补充方法利用了核酸酶的 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 调节基因组调控区域的转录状态，例如启动子或增强子，而无需 DSB 的需要。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 有效编辑 HSC 以阐明造血与 CVD 之间的联系。主要目标 核心 2 包括通过优化实验方法来支持本提案的项目，并提供 项目 1 至 4 的关键实验的基因组和表观基因组编辑试剂和专业知识。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。具体来说，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 HSC 中实现高效、特异的基因组编辑策略 用于 HSC 的编辑试剂，以及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 编辑的成功优化和开发将加快 项目 1 至 4 检查造血和 CVD 之间的关系以及更广泛的能力， 这些进展将广泛应用于心血管和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.