Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells

项目总结/摘要。在理解CRISPR- 基因编辑器在不同的人类细胞中产生基因修饰。缺乏对原因的理解 不同的编辑器失败了，为什么有些编辑器成功地创建了所需的基因编辑-同时保留了完整的细胞和组织 功能-限制基因组编辑工具的使用。通过实时观察患者体内的基因组编辑- 在体外衍生细胞，我试图了解在人类细胞上进行基因组编辑的瓶颈， 精确控制的基因组编辑器粒子。粒子将与各种DNA，RNA， 和聚合物组分并递送至患者来源的细胞和微组织。原位高容量成像 定制细胞基质内的分析将在多个尺度上监测基因组编辑。中央 一种假设是，CRISPR-Cas9颗粒的新组装体可以探测不同的生物过程， 运输，DNA双链断裂形成和DNA修复参与人类细胞的基因组编辑 以及对涉及细胞周期停滞的生物过程的下游影响， 形态发生这一假设将在患者来源的干细胞和组织中进行测试，以检测两种基因的表达。 破坏和纠正。拟议研究的一个总体理由是， 与基因组编辑有关的基本生物学过程可以使新细胞的开发成为可能。 用于未来基因组和精准医学的治疗和基因治疗。以强大的生产力为指导， 目前的早期阶段R35奖，我将从事三个研究计划：1）组装Cas9颗粒，以确定 促进基因校正的人类细胞内的染色质结构; 2）组装Cas9颗粒以鉴定 促进干细胞内基因校正的递送和DNA修复过程;以及，3）组装Cas9 颗粒，以鉴定促进基因校正的细胞增殖和组织形态发生过程， 患者来源的微组织中的病变突变。根据第一个研究计划，编辑将发生在目标 诱导多能干细胞（iPSC）内具有可变染色质结构的基因， 后代，并与小分子治疗。在第二和第三个研究计划中，基因组编辑 将应用于基因校正iPSC中的患病突变，以及由它们成熟的微组织。的方法 在申请人看来是创新的，因为它通过系统地改变现状， 在患者来源的细胞中使用新方法一次多个组分。所提出的研究是有意义的 因为它有望推进和扩大我们对基因组编辑工具如何应用的理解， 用于产生从靶向小分子到细胞/组织疗法的先进疗法。 最终，这些知识将为涉及基因组编辑的新翻译项目奠定基础。

PROJECT SUMMARY/ ABSTRACT. There continues to be a fundamental gap in understanding how CRISPR- based genome editors produce gene modifications in different human cells. A lack of understanding of why various editors fail and why some succeed in creating desired gene edits - while retaining full cell and tissue functionality - limits the use of genome editing tools. By observing genome editing in real-time within patient- derived cells in vitro, I seek to understand the bottlenecks in performing genome editing on human cells with precisely-controlled genome editor particles. Particles will be systematically assembled with various DNA, RNA, and polymeric components and delivered to patient-derived cells and microtissues. In situ high content imaging and analysis within customized cell substrates will monitor genome editing at multiple scales. The central hypothesis is that new assemblies of CRISPR-Cas9 particles can probe different biological processes of trafficking, DNA-double strand break formation, and DNA repair involved in the genome editing of human cells and tissues, as well as downstream effects on biological processes involving cell cycle arrest and morphogenesis. This hypothesis will be tested within patient-derived stem cells and tissues for both gene disruption and correction. An overarching rationale for the proposed research is that an improved understanding of fundamental biological processes involved with genome editing could enable the development of novel cell therapies and gene therapies for future genomic and precision medicine. Guided by strong productivity in the current early stage R35 award, I will pursue three research programs: 1) Assemble Cas9 particles to identify chromatin structures within human cells that promote gene correction; 2) Assemble Cas9 particles to identify delivery and DNA repair processes that promote gene correction within stem cells; and, 3) Assemble Cas9 particles to identify cell proliferative and tissue morphogenesis processes that promote gene correction of diseased mutations in patient-derived microtissues. Under the first research program, editing will occur at target genes that have variable chromatin structures within induced pluripotent stem cells (iPSCs), differentiated progeny, and with small-molecule treatment. Under the second and third research programs, genome editors will be applied to gene-correct diseased mutations in iPSCs, and microtissues matured from them. The approach is innovative, in the applicant’s opinion, because it departs from the status quo by systematically changing multiple components at a time using novel methods in patient-derived cells. The proposed research is significant because it is expected to advance and expand our understanding of how genome editing tools can be applied for the generation of advanced therapeutics, ranging from targeted small molecules to cell/tissue therapies. Ultimately, such knowledge would solidify the foundation for new translational projects involving genome editing.