Uncovering cell-intrinsic restrictions to CRISPR-Cas9 gene editing

揭示 CRISPR-Cas9 基因编辑的细胞内在限制

• 批准号: 10596613
• 负责人: Jennifer R Hamilton
• 金额: $ 11.82万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2023-10-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10596613
• 关键词: Address; Area; Award; Biological Models; Biological Process; Biology; Bone Marrow; COVID-19; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cell Survival; Cell model; Cell physiology; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; DNA; DNA Damage; DNA Repair; DNA Sequence; Development; Disease; Down-Regulation; Engraftment; Environment; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genomics; Goals; Hematopoietic; Hematopoietic stem cells; Homeostasis; Human; In Situ; Infusion procedures; International; Intrinsic factor; Knowledge; Link; Longevity; Maintenance; Mammalian Cell; Mediating; Mentors; Modeling; Modification; Mus; Outcome; Phase; Physiological; Postdoctoral Fellow; Predisposition; Primary Cell Cultures; Regulator Genes; Research; Ribonucleoproteins; Running; San Francisco; Site; T cell differentiation; TP53 gene; Technology; Testing; Tissues; Training; Treatment Efficacy; Undifferentiated; Virus-like particle; Work; adult stem cell; base editing; cell type; comparative effectiveness; delivery vehicle; experience; experimental study; genetic manipulation; genome editing; genome-wide; improved; improved outcome; in vivo; innovation; insight; multipotent cell; new technology; response; single-cell RNA sequencing; somatic cell gene editing; stem cell biology; stem cell homeostasis; stem cells; symposium; targeted treatment; therapeutic gene; therapeutic genome editing; transcriptional reprogramming; transcriptome; translational potential

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.

项目摘要 多能性、组织特异性干细胞（“成体干细胞”）是治疗性基因编辑的主要靶点 因为它们的寿命和分化成专门细胞类型的能力。然而，网站导向 成体干细胞的遗传修饰在体内是低效的。此外，没有对基因组的稳健表征 已经进行了跨完整细胞谱系的编辑效率以理解细胞内在限制 到未分化和分化细胞类型中的基因编辑。我的大部分博士后工作都集中在 开发病毒样颗粒作为预组装CRISPR Cas9-sgRNA核糖核蛋白的递送载体 (RNP)用于离体编辑原代人细胞的复合物。因此，本建议的主要目的是：1） 为了表征来源于细胞的细胞类型的基线相对基因编辑和碱基编辑效率， 含有多能和分化细胞类型的完整细胞谱系（造血细胞），2）杠杆作用 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 为了鉴定调节基因编辑效率的遗传因子，3）将CRISPR-i和Perturb-seq偶联， 揭示基因编辑后负责维持成体干细胞稳态的遗传因素，以及4） 利用包装Cas9 RNP复合物的病毒样颗粒实现成体干细胞的基因组编辑， vivo.这项拟议中的研究将为基因编辑的基础生物学提供相当多的见解 未分化和分化的原代细胞中的决定子，以及使用Cas9 RNP来 使用造血干细胞作为模型细胞，在体内介导多能细胞中的治疗性基因编辑 类型.与干细胞生物学、DNA修复生物学、治疗基因组学领域相关的重大发现 编辑期待。其他科学培训领域，将使成功完成这一目标 建议是原代细胞培养的知识，进行全基因组CRISPR-Cas筛选的经验 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都位于 在加州大学伯克利分校或在大旧金山弗朗西斯科湾区科学界。在指导阶段， 在这个项目中，我将继续开展科学专业发展活动，以提高作为科学领导者的水平， 获得了一个全面的基础，在主题至关重要的运行我自己的独立研究小组。我也会 继续在国家和国际会议上展示我的研究（可能是远程的，而COVID-19 预防措施有效）。当与加州大学伯克利分校的优秀研究环境和 创新基因组学研究所，我有一个很好的机会来完成我的基础训练，因为我 开始我独立的过渡。

项目成果

In vivo human T cell engineering with enveloped delivery vehicles.

• DOI: 10.1038/s41587-023-02085-z
• 发表时间: 2024-01
• 期刊: Nature biotechnology
• 影响因子: 46.9
• 作者: J. Hamilton;Evelyn Chen;Barbara S. Perez;Cindy R Sandoval Espinoza;Min Hyung Kang;M. Trinidad;Wayne Ngo;Jennifer A. Doudna