Mechanism and therapeutic application of RNA-guided immune systems

 描述（由申请人提供）：在动物和植物中使用细菌RNA引导的CRISPR-Cas9免疫系统的基因组工程正在改变生物学。从其发现到阐明酶机制的努力为在各种细胞和生物体中修饰、调节或标记基因组位点的显着发展提供了基础。CRISPR-Cas9基因编辑有可能通过提供创新的方法来探索生物学和治疗成人遗传疾病来改变医学。该项目的主要目标是：1）在生物化学和结构生物学的新领域进行培训（K99阶段），2）表征和开发用于生物医学应用的新型Cas9变体（K99/R 00阶段），以及3）在领先的学术机构建立一个成功的独立研究实验室（R 00阶段）。我的实验室将专注于RNA引导的免疫系统的机制和治疗应用，以解决对新平台技术的迫切医疗需求，以建立多样化的治疗靶点和开发创新的治疗模式，并指导下一代科学家。候选人：我致力于生物医学研究的学术生涯。我的长期目标是在一个领先的学术机构获得终身教职，并成功建立一个独立的研究实验室。我的工作重点是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体内模型方面的多学科培训，结合在K99指导阶段学到的新的生物化学和结构方法，将使我能够弥合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的治疗模式。重要的是，我致力于指导下一代学生，并作为榜样，帮助有才华的年轻科学家转化为成功的教师。环境：指导阶段（K99）将在Jennifer A博士的实验室进行。Doudna在加州大学伯克利分校，一个高度互动和充满活力的研究环境，学习生物化学和结构生物学的新技能。Doudna实验室位于斯坦利大厅，作为加州大学伯克利分校的中心，为加州定量生物科学研究所（QB 3）。Doudna实验室与QB 3的200多名研究人员进行了许多多学科的互动，涉及生物科学，化学科学，物理科学和工程学。这些合作导致更好地获得生物化学，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方面的尖端专业知识。我将受益于这一巨大的人才和知识资源，以及获得开展拟议研究所需的专门设备。重要的是，Doudna博士是生物化学和结构生物学的领先专家，也是CRISPR-Cas9基因组编辑技术的先驱。调研：CRISPR-Cas9基因编辑有可能通过提供创新的方法来探测生物学和治疗成人患者的遗传疾病来增强医学。该项目的目标是定义机制并建立新的Cas9变体，用于有效的CRISPR-Cas9介导的体内基因组编辑。为此，我们将结合联合收割机计算，生物化学和高通量细胞为基础的方法。该提案的具体目的是：目的1）表征新型Cas9内切核酸酶，以及目的2）建立用于有效体内基因组编辑的DNA引导的CRISPR系统。拟议项目的完成将产生新的平台技术和应用，用于高效的基于CRISPR的基因编辑，以满足对新工具的迫切医疗需求，以加速药物发现和开发创新的治疗模式。1)新定义的Cas9酶将产生正交CRISPR系统，可并行用于多重基因组编辑。这些工具将允许以前所未有的精度和速度探索生物学，并扩大我们对稳态和疾病的理解。反过来，这将为患者提供更好的治疗。此外，靶向Cas9的ssDNA或ss/dsRNA可能导致评估基因组/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CRISPR的创新策略以治疗遗传疾病的基础。重要的是，在该奖项的指导阶段（K99）学到的方法和生成的数据将为我提供未来项目和赠款（R 00及以后）的基础。加上我在细胞生物学和动物模型方面的专业知识，这将使我能够成功地在一家领先的学术研究机构建立一个独立的实验室。

 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.