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分如何 在人类细胞中实现基因修饰。这一差距的继续存在是一个重要问题 因为，在它被填满之前，基因组手术工具的使用将受到限制，因为不清楚为什么各种核酸酶失败， 以及为什么有些人成功地产生了所需的基因编辑。长期目标是观察基因组手术， 行动，以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组分组成。粒子将系统地 其与各种组分组装并以受控方式递送至患者来源的细胞和组织。 在定制的细胞基质内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组分的大序列变异，沿着出现新的CRISPR-Cas9突变。 CRISPR-Cas9组件的组装。核心假设是CRISPR-Cas9的新组装 颗粒可以探测不同的生物过程的运输，DNA双链断裂形成和DNA 基因组手术中的修复。这一假设将被测试方面产生两种类型的基因 涉及非同源末端连接（NHEJ）和同源定向修复（HDR）途径的编辑， 患者来源的干细胞和组织内的基因组基因座。拟议研究的总体理由 强大的基因编辑技术可以使个性化药物，细胞 用于未来基因组和精准医学的治疗和基因治疗。在强有力的初步数据的指导下， 这一假设将通过以下三个研究项目进行验证：1）组装Cas9颗粒， 促进培养物中干细胞命运的“无标记”转录物标记的生物学过程; 2）组装 Cas9颗粒用于鉴定促进干细胞中患病突变的基因校正的生物过程 3）组装Cas9颗粒以鉴定促进细胞的基因校正的生物过程， 微组织中的病变突变在第一个研究计划下，一个已经被证明的平台， 数百种独特的Cas9颗粒和以多重方式编辑患者来源的细胞，将用于 监测NHEJ在干细胞标记基因内产生的小基因编辑。在第二和第三 研究计划，该平台将应用于通过HDR诱导的基因校正疾病突变。 多能干细胞和由其成熟的微组织。该方法是创新的，在申请人的 因为它通过一次系统地改变多个组成部分而脱离现状 使用新的方法在患者来源的细胞中。这项研究意义重大，因为它有望 推进和扩大对基因组手术工具如何应用于产生 先进的治疗方法，从靶向小分子到自体细胞疗法。最终，这样的 知识有可能为精准医学的新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.