Mechanism and therapeutic application of RNA-guided immune systems

RNA引导的免疫系统的机制和治疗应用

• 批准号: 9306142
• 负责人: Christof Fellmann
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306142
• 关键词: Address; Adult; Animal Model; Animals; Award; Bacterial RNA; Basic Science; Binding; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Biological Sciences; Biology; Biomedical Research; Biophysics; CRISPR/Cas technology; California; Cell Culture Techniques; Cells; Cellular biology; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Computer Simulation; Crystallization; DNA; Data; Data Analyses; Development; Disease; Double-Stranded RNA; Engineering; Environment; Enzymes; Equipment; Eukaryotic Cell; Faculty; Fluorescence; Foundations; Future; Gene Silencing; Genes; Genome; Genome engineering; Genomics; Goals; Grant; Guide RNA; Half-Life; Hereditary Disease; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Immune system; Immunity; Institutes; Institution; Knowledge; Laboratories; Laboratory Research; Lead; Learning; Libraries; Malignant Neoplasms; Mammalian Cell; Mediating; Medical; Medicine; Mentors; Methods; MicroRNAs; Modality; Modification; Molecular; Mutation; Nonhomologous DNA End Joining; Organism; Patients; Phase; Plants; Positioning Attribute; RNA; RNA Interference; RNA Phages; Research; Research Personnel; Resources; Ribonucleoproteins; Safety; Science; Scientist; Secure; Serum; Small Interfering RNA; Speed; Streptococcus pyogenes; Structure; Students; System; Talents; Technology; Testing; Therapeutic; Therapeutic Uses; Training; Variant; Vertebral column; Viral Vector; Work; base; career; combinatorial; deep sequencing; design; drug discovery; ds-DNA; endonuclease; enzyme mechanism; genome editing; human disease; in vivo; in vivo Model; innovation; insight; member; multidisciplinary; mutant; next generation; novel; physical science; public health relevance; repaired; restriction enzyme; role model; sensor; skills; small hairpin RNA; structural biology; targeted treatment; tenure track; therapeutic target; tool; transcriptome; translational approach; tumorigenesis

 DESCRIPTION (provided by applicant): Genome engineering using the bacterial RNA-guided CRISPR-Cas9 immune system in animals and plants is transforming biology. Efforts from its discovery through the elucidation of the enzyme mechanism are providing the foundation for remarkable developments to modify, regulate or mark genomic loci in a wide variety of cells and organisms. CRISPR-Cas9 gene editing has the potential to transform medicine by providing innovative ways to probe biology and treat genetic disorders in adults. The key goals of the proposed project are: 1) to train in the new fields of biochemistry and structural biology (K99 phase), 2) to characterize and develop novel Cas9 variants for biomedical applications (K99/R00 phase), and 3) to establish a successful, independent research laboratory at a leading academic institution (R00 phase). My laboratory will be focused on mechanism and therapeutic application of RNA-guided immune systems, to address the urgent medical need for new platform technologies to establish diverse therapeutic targets and develop innovative treatment modalities, and to mentor the next generation of scientists. Candidate: I am committed to an academic career in biomedical research. My long-term goals are to secure a tenure-track faculty position at a leading academic institution and successfully establish an independent research laboratory. My work focuses on mechanism and therapeutic application of RNA-guided immune systems. My multidisciplinary training in molecular cell biology and in-vivo models of human disease, combined with the new biochemical and structural approaches learned during the K99 mentored phase, will allow me to bridge basic science and patient-focused research. In turn, this will allow me to help accelerate translational approaches and develop novel treatment paradigms for genetic disorders. Importantly, I am committed to mentor the next generation of students and serve as a role model to help transform talented young scientist into successful faculty members. Environment: The mentored phase (K99) will be carried out in the laboratory of Dr. Jennifer A. Doudna at UC Berkeley, a highly interactive and vibrant research environment, to learn new skills in biochemistry and structural biology. The Doudna Laboratory is located at Stanley Hall, which serves as the UC Berkeley hub for the California Institute for Quantitative Bioscience (QB3). The Doudna lab has many multidisciplinary interactions with the more than 200 researchers that are part of QB3, involving the the biological sciences, chemical sciences, physical sciences and engineering. These collaborations result in enhanced access to cutting-edge expertise in biochemistry, structural biology, biophysics, computational modeling, high-throughput sequencing and large-scale data analysis. I will benefit from this vast resource of talent and knowledge, as well as access to the specialized equipment needed to carry out the proposed research. Importantly, Dr. Doudna is a leading expert in biochemistry and structural biology, and a pioneer of the CRISPR-Cas9 genome editing technology. Research: CRISPR-Cas9 gene editing has the potential to enhance medicine by providing innovative ways to probe biology and treat genetic disorders in adult patients. The goal of this project is to define mechanisms and establish novel Cas9 variants for efficient CRISPR-Cas9 mediated genome editing in vivo. To this end, we will combine computational, biochemical and high-throughput cell-based approaches. The Specific Aims of this proposal are: Aim 1) to characterize novel Cas9 endonucleases, and Aim 2) to establish a DNA-guided CRISPR system for efficient in-vivo genome editing. Completion of the proposed project will result in new platform technologies and applications for efficient CRISPR-based gene editing, to meet the urgent medical need for new tools to accelerate drug discovery and develop innovative treatment modalities. 1) Newly defined Cas9 enzymes will yield orthogonal CRISPR systems that can be used in parallel for multiplexed genome editing. These tools will allow probing biology with unprecedented precision and speed, and expand our understanding of homeostasis and disease. In turn, this will lead to better treatments for patients. Additionally, ssDNA or ss/dsRNA targeting Cas9s may lead to new methods to assess genomes/transcriptomes and can provide novel insight into the mechanisms of bacterial immunity. 2) The DNA-guided Cas9 version will facilitate therapeutic applications by overcoming the bottleneck of sgRNA instability in serum, and constitute the basis for the development of innovative CRISPR-based strategies to treat genetic disorders. Importantly, the methods learned and data generated during the mentored phase of the award (K99) will provide me with the foundation for future projects and grants (R00 and beyond). Together with my expertise in cell biology and animal modeling, this will allow me to successfully establish an independent laboratory at a leading academic research institution.

 描述（应用程序提供）：使用细菌RNA引导的CRISPR-CAS9免疫系统中的基因组工程在动物和植物中正在转化生物学。通过阐明酶机制发现的努力为在各种细胞和生物中修改，调节或标记基因组局部的非凡发展为基础提供了基础。 CRISPR-CAS9基因编辑有可能通过提供创新的方法来探究生物学和治疗成人遗传疾病的方法。拟议项目的主要目标是：1）在新的生物化学和结构生物学（K99阶段）中训练，2）表征和开发用于生物医学应用的新颖CAS9变体（K99/R00阶段），以及3），以在领先的学术机构（R00阶段）建立成功的独立研究实验室（R00阶段）。我的实验室将重点介绍RNA引导的免疫系统的机制和治疗应用，以应对对新平台技术的紧急医疗需求，以建立多样性治疗目标并发展创新的治疗方式，并使下一代科学家进行心理心理。候选人：我致力于生物医学研究的学术生涯。我的长期目标是在领先的学术机构中确保终身任职的教师职位，并成功建立一个独立的研究实验室。我的工作着重于RNA引导的免疫系统的机制和治疗应用。我在分子细胞生物学和人类疾病的体内模型中进行的多学科培训，再加上在K99修正阶段学习的新生化和结构方法，将使我能够桥接基础科学和以患者为中心的研究。反过来，这将使我能够帮助加速转化方法并为遗传疾病开发新颖的治疗范例。重要的是，我致力于指导下一代学生，并作为榜样，以帮助将才华横溢的年轻科学家转变为成功的教师。环境：修订阶段（K99）将在加州大学伯克利分校的Jennifer A. Doudna博士的实验室中进行，这是一个高度交互式且充满活力的研究环境，以学习生物化学和结构生物学方面的新技能。 Doudna实验室位于Stanley Hall，后者是加利福尼亚定量生物科学研究所的UC伯克利枢纽（QB3）。 Doudna实验室与QB3的200多名研究人员有许多跨学科的互动，涉及生物学，化学科学，物理科学和工程。这些合作导致增强了对生物化学，结构生物学，生物物理学，计算建模，高通量测序和大规模数据分析的尖端专业知识的访问。我将从这种庞大的人才和知识资源中受益，以及对进行拟议研究所需的专业设备的访问。重要的是，Doudna博士是生物化学和结构生物学领域的领先专家，也是CRISPR-CAS9基因组编辑技术的先驱。研究：CRISPR-CAS9基因编辑有可能通过提供创新的方法来探测成人患者的生物学和治疗遗传疾病的方法。该项目的目的是定义机制并建立新颖的CAS9变体，以在体内有效的CRISPR-CAS9介导的基因组编辑。为此，我们将结合计算，生化和高通量细胞的方法。该提案的具体目的是：目标1）表征新颖的Cas9核酸内切酶，目标2）建立一个DNA引导的CRISPR系统，以有效的体内基因组编辑。拟议项目的完成将为有效的基于CRISPR的基因编辑提供新的平台技术和应用程序，以满足对加速药物发现并开发创新治疗方式的新工具的紧急医疗需求。 1）新定义的CAS9酶将产生可行的多路复用基因组编辑的正交CRISPR系统。这些工具将允许以空前的精度和速度进行探测生物学，并扩展我们对体内稳态和疾病的理解。反过来，这将为患者提供更好的治疗方法。此外，针对Cas9的ssDNA或SS/DSRNA可能会导致评估基因组/转录组的新方法，并可以提供对细菌免疫机制的新见解。 2）DNA引导的CAS9版本将通过克服血清中SGRNA不稳定性的瓶颈来促进治疗应用，并构成开发基于创新的CRISPR治疗遗传疾病的策略的基础。重要的是，在奖励的修订阶段（K99）中学到的方法和数据将为我提供未来项目和赠款的基础（R00及以后）。以及我在细胞生物学和动物建模方面的专业知识，这将使我能够在领先的学术研究机构成功建立独立的实验室。