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

项目摘要/摘要。在理解CRISPR如何-- 基于基因组的编辑在不同的人类细胞中产生基因修改。缺乏对原因的理解 不同的编辑者失败了，为什么有些编辑者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，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粒子。粒子将由不同的DNA，RNA， 和聚合物组分，并输送到患者来源的细胞和微组织。原位高含量成像 定制细胞底物中的分析将在多个尺度上监控基因组编辑。中环 假设CRISPR-Cas9颗粒的新组装可以探测不同的生物学过程 参与人类细胞基因组编辑的运输、DNA双链断裂形成和DNA修复 以及对生物过程的下游影响，包括细胞周期停滞和 形态发生。这一假设将在患者来源的干细胞和组织中进行验证，以检测这两种基因 颠覆和修正。拟议研究的一个主要理由是，更好的理解 与基因组编辑相关的基本生物学过程的研究可以使新细胞的发展成为可能 未来基因组和精确医学的疗法和基因疗法。在强劲的生产力指导下， 目前R35早期奖，我将从事三个研究项目：1)组装Cas9颗粒以识别 人类细胞内促进基因校正的染色质结构；2)组装Cas9颗粒以识别 促进干细胞内基因纠正的传递和DNA修复过程；以及，3)组装Cas9 用于识别促进基因校正的细胞增殖和组织形态发生过程的颗粒 病人来源的微组织中的病态突变。根据第一个研究计划，编辑将在目标位置进行 诱导多能干细胞(IPSCs)内具有可变染色质结构的分化基因 子代，并进行小分子治疗。在第二和第三个研究计划下，基因组编辑 将被应用于IPSCs中的基因纠正疾病突变，并从这些突变中成熟出微组织。该方法 在申请人看来，这是创新的，因为它通过系统地改变 在患者来源的细胞中使用新的方法一次使用多种成分。这项拟议的研究具有重要意义 因为它有望促进和扩大我们对如何应用基因组编辑工具的理解 用于产生先进的治疗方法，从靶向小分子到细胞/组织治疗。 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.