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; 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 多能组织特异性干细胞（“成年干细胞”）是治疗基因编辑的主要靶标 由于它们的寿命和能力分为专门的细胞类型。但是，该站点定向 成年干细胞的遗传修饰在体内效率低下。此外，没有基因组的鲁棒表征 已经执行了整个细胞谱系的编辑效率，以了解细胞中的限制 在未分化和分化的细胞类型中进行基因编辑。我的大部分博士后工作都集中在 开发类似病毒的颗粒作为预组装CRISPR CAS9-SGRNA Ribbanuleoprotoin的递送工具 （RNP）用于编辑原代人细胞的复合物。因此，该提案的主要目的是：1） 为了表征基线相对基因编辑和基础编辑效率的细胞类型的效率 完整的细胞谱系（造血细胞），包含多能和分化的细胞类型，2）杠杆作用 成年干细胞（造血干细胞）和终末分化细胞（T细胞）中的CRISPR-CAS筛选 要确定调节基因编辑效率的遗传因素，3）将CRISPR-I和witturb-seq逐渐 发现基因编辑后负责维持成年干细胞稳态的遗传因素，4） 利用病毒样颗粒包装CAS9 RNP复合物来实现成年干细胞的基因组编辑 体内。拟议的研究将为基本生物学的基础基因编辑提供考虑的洞察力 未分化和分化的原代细胞中的决定因素，以及使用Cas9 RNP的可行性 使用造血干细胞作为模型细胞中的多能细胞中的介导疗法基因编辑 类型。与干细胞生物学，DNA修复生物学，治疗基因组有关的重要发现 预期编辑。其他科学培训的领域将成功完成 建议是对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。该奖项的修订阶段将由詹妮弗·杜德纳（Jennifer Doudna）博士进行监督 基因组编辑技术领域的世界领导者。 Doudna博士和该项目的所有其他合作者都位于 在加州大学伯克利分校或大旧金山湾地区科学界。在指导阶段 这个项目我将继续科学专业发展活动，以改善科学领导者和 在管理自己的独立研究小组至关重要的主题中获得彻底的基础。我也会 继续在国家和国际会议上介绍我的研究（很可能是远程COVID-19 预防措施实际上是）。当与加州大学伯克利分校的出色研究环境结合使用时 创新的基因组学研究所，我有一个很棒的机会来完成我的基础培训 开始我向独立过渡。