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

在理解工程基因编辑核酸酶的几个组成部分如何 在人类细胞中实现基因修饰。这种差距的持续存在是一个重要的问题 因为，在它被填满之前，基因组手术工具的使用将受到限制，因为尚不清楚各种核酸酶失败的原因。 以及为什么有些人成功地产生了所需的基因编辑。长期目标是观察中国的基因组手术 了解在体外对人类细胞进行基因组手术的瓶颈 受控基因编辑颗粒，由CRISPR-Cas9组件组成。粒子将被系统地 用各种成分组装，并以受控方式输送到患者来源的细胞和组织。 在定制的细胞底物中进行现场高含量成像和分析将监测基因组手术。 这些能力将探索CRISPR-Cas9组件的大序列变异以及新的 CRISPR-CAS9组件的装配。中心假设是CRISPR-Cas9的新组件 粒子可以探测运输、DNA双链断裂形成和DNA的不同生物过程 修复涉及到基因组手术。这一假设将通过产生两种类型的基因来检验。 编辑涉及多个非同源末端连接(NHEJ)和同源定向修复(HDR)途径 患者来源的干细胞和组织中的基因组位置。拟议研究的最重要的理由 程序是强大的基因编辑技术可以使生产个性化药物，细胞 未来基因组和精确医学的疗法和基因疗法。在强劲的初步数据的指引下， 这一假设将通过三个研究项目得到验证：1)组装Cas9粒子以识别 促进干细胞在培养中命运的“无报告”转录本标记的生物过程；2)组装 Cas9颗粒用于识别促进茎中患病突变的基因纠正的生物过程 细胞；以及，3)组装Cas9颗粒以识别促进基因校正的生物学过程 微小组织中的病态突变。在第一个研究计划下，一个已经得到验证的平台，以组装 数百个独特的Cas9颗粒和以多路复用方式编辑患者来源的细胞将被用于 监测NHEJ在干细胞标记基因内的小基因编辑的产生。在第二个和第三个下面 研究计划，该平台将应用于通过HDR诱导的基因纠正疾病突变 多能干细胞和微组织由它们成熟而来。这种方法是创新的，在申请人的 意见，因为它通过一次系统地改变多个组件来偏离现状 在患者来源的细胞中使用新方法。这项拟议的研究意义重大，因为预计它将 促进和扩大对基因组外科工具如何应用于产生 先进的疗法，从靶向小分子到自体细胞疗法。最终，这样的 知识有可能为精密医学的新的临床前平台奠定基础。

There is a fundamental gap in understanding how several components of engineered gene-editing nucleases achieve gene modification in human cells. Continued existence of this gap represents an important problem because, until it is filled, use of genome surgery tools will be limited, as it is not clear why various nucleases fail and why some succeed in producing desired gene edits. The long-term goal is to watch genome surgery in action to understand the bottlenecks in performing genome surgery on human cells in vitro with precisely controlled gene-editing particles, comprised of CRISPR-Cas9 components. Particles will be systematically assembled with various components and delivered in a controlled fashion to patient-derived cells and tissues. Live, in situ high content imaging and analysis within customized cell substrates will monitor genome surgery. These capabilities will explore large sequence variation of CRISPR-Cas9 components along with new assemblies of CRISPR-Cas9 components. 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 genome surgery. This hypothesis will be tested with respect to generating two types of gene edits involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways at several genomic loci within patient-derived stem cells and tissues. An overarching rationale for the proposed research programs is that robust gene editing techniques could enable the production of personalized drugs, cell therapies and gene therapies for future genomic and precision medicine. Guided by strong preliminary data, this hypothesis will be tested by pursuing three research programs: 1) Assemble Cas9 particles to identify biological processes that promote "reporter-less" transcript tagging of stem cell fate in culture; 2) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in stem cells; and, 3) Assemble Cas9 particles to identify biological processes that promote gene correction of diseased mutations in microtissues. Under the first research program, an already proven platform, to assemble hundreds of unique Cas9 particles and edit patient-derived cells in a multiplexed manner, will be used to monitor the production of small gene edits by NHEJ within stem cell marker genes. Under the second and third research programs, this platform will be applied to gene-correct diseased mutations via HDR in induced pluripotent stem cells 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 understanding of how genome surgery tools can be applied for the generation of advanced therapeutics, ranging from targeted small molecules to autologous cell therapies. Ultimately, such knowledge has the potential to set the foundation for new preclinical platforms in Precision Medicine.