Uncovering cell-intrinsic restrictions to CRISPR-Cas9 gene editing

项目摘要 多能、组织特异性干细胞(“成体干细胞”)是治疗性基因编辑的主要目标。 因为它们的寿命和分化成特殊细胞类型的能力。然而，该网站定向的 成体干细胞的遗传修饰在体内是低效的。此外，没有强有力的基因组特征 对整个细胞谱系的编辑效率进行了测试，以了解细胞的内在限制 到未分化和已分化细胞类型的基因编辑。我的博士后工作主要集中在 发展病毒样颗粒作为预组装CRISPR Cas9-sgRNA核糖核蛋白的载体 (RNP)复合体，用于体外编辑原代人类细胞。因此，这项建议的主要目的是：1) 为了表征起源于 包含多能和分化细胞类型的完整细胞谱系(造血细胞)，2)杠杆 CRISPR-Cas在成体干细胞(造血干细胞)和终末分化细胞(T细胞)中的筛选 为了确定调节基因编辑效率的遗传因素，3)将CRISPR-I和扰动序列连接到 发现基因编辑后维持成人干细胞稳态的遗传因素和4) 利用病毒样颗粒包装Cas9 RNP复合体实现成体干细胞基因组编辑 活着。这项拟议的研究将为基因编辑的基础生物学提供相当大的洞察力 未分化和已分化原代细胞中的决定因素，以及使用Cas9 RNPs治疗 以造血干细胞为模型细胞的体内多能细胞介导性治疗性基因编辑 打字。与干细胞生物学、DNA修复生物学、治疗基因组领域相关的重大发现 预计将进行编辑。能够顺利完成这项工作的额外科学培训领域 建议包括原代细胞培养知识、进行全基因组CRISPR-CAS筛查经验 和单细胞RNAseq分析。该奖项的指导阶段将由詹妮弗·杜德纳博士监督， 基因组编辑技术的世界领先者。杜德纳博士和这个项目的所有其他合作者 在加州大学伯克利分校或旧金山湾区科学界。在指导阶段 本项目我将继续开展科学职业发展活动，以提高作为科学带头人的地位 对运营我自己的独立研究小组所必需的主题有一个透彻的基础。我也会 继续在国内和国际会议上发表我的研究成果(可能在新冠肺炎上远程发表 预防措施已经生效)。与加州大学伯克利分校出色的研究环境和 创新基因组学研究所，我有一个绝佳的机会来完成我的基础培训 开始我向独立的过渡。

Project Summary Multipotent, tissue-specific stem cells (“adult stem cells”) are major targets for therapeutic gene editing because of their longevity and capacity to differentiate into specialized cell types. However, the site-directed genetic modification of adult stem cells is inefficient in vivo. Further, no robust characterization of genome editing efficiency across a complete cellular lineage has been performed to understand cell-intrinsic restrictions to gene editing in undifferentiated and differentiated cell types. Much of my postdoctoral work has focused on developing virus-like particles as a delivery vehicle for pre-assembled CRISPR Cas9-sgRNA ribonucleoprotein (RNP) complexes for editing of primary human cells ex vivo. The primary aims of this proposal are therefore: 1) to characterize the the baseline relative gene editing and base editing efficiencies of cell types derived from a complete cell lineage (hematopoietic cells) containing multipotent and differentiated cell types, 2) leverage CRISPR-Cas screens in adult stem cells (hematopoietic stem cells) and terminally differentiated cells (T cells) to identify genetic factors that modulate gene editing efficiency, 3) couple CRISPR-i and Perturb-seq to uncover genetic factors responsible for maintaining adult stem cell homeostasis following gene editing and 4) utilize virus-like particles packaging Cas9 RNP complexes to achieve genome editing of adult stem cells in vivo. The proposed research will provide considerable insight into the basic biology underpinning gene editing determinants in undifferentiated and differentiated primary cells, and the feasibility of using Cas9 RNPs to mediate therapeutic gene editing in multipotent cells in vivo, using hematopoietic stem cells as a model cell type. Significant findings relevant to the fields of stem cell biology, DNA repair biology, therapeutic genome editing are expected. Areas of additional scientific training that will enable successful completion of this proposal are knowledge of primary cell culture, experience conducting genome-wide CRISPR-Cas screens and single-cell RNAseq analysis. The mentored phase of the award will be supervised by Dr. Jennifer Doudna, a world-leader in genome editing technology. Dr. Doudna, and all other collaborators on this project are located at UC Berkeley or in the greater San Francisco Bay Area scientific community. During the mentored phase of this project I will continue scientific professional development activities to improve as a scientific leader and gain a thorough grounding in topics essential for running my own independent research group. I will also continue presenting my research at national and international conferences (likely remotely while COVID-19 precautions are in effect). When combined with the excellent research environment at UC Berkeley and the Innovative Genomics Institute, I have an outstanding opportunity to complete my foundational training as I begin my transition to independence.