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: 10449495
• 负责人: Jennifer R Hamilton
• 金额: $ 9.57万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449495
• 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; Foundations; Gene Expression Profile; Genes; Genetic; Genetic Determinism; Genomics; Goals; Hematopoietic; Hematopoietic stem cells; Homeostasis; Human; In Situ; Infusion procedures; Institutes; International; Intrinsic factor; Knowledge; Link; Longevity; Maintenance; Mammalian Cell; Mediating; Mentors; Modeling; Modification; Mus; Outcome; Phase; Physiological; Predisposition; Primary Cell Cultures; Regulator Genes; Research; Ribonucleoproteins; Running; San Francisco; Site; Supervision; T cell differentiation; T-Lymphocyte; 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 genes; 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.

项目总结