In vivo functional screen of noncoding genetic elements

非编码遗传元件的体内功能筛选

• 批准号: 10507140
• 负责人: Saba Parvez
• 金额: $ 10.11万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-02 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507140
• 关键词: Animal Model; Animals; Behavior; Biochemical; Bioinformatics; Biological Assay; Biological Process; Brain; Budgets; CRISPR interference; CRISPR/Cas technology; Cell Culture Techniques; Cell Proliferation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Communities; Complement; Complex; Development; Developmental Biology; Disease; Elements; Embryo; Embryonic Development; Enhancers; Ensure; Evaluation; Exclusion; Exons; Eye Development; Five-Year Plans; Genes; Genetic; Genetic Screening; Genome; Genomic Segment; Genomics; Growth; Guide RNA; Health; Human; Human Genome; Human Genome Project; Immunity; In Vitro; Infection; Injections; Knock-out; Knowledge; Link; Mammalian Cell; Mentors; Mentorship; Modeling; Molecular Profiling; Neoplasm Metastasis; Neurodevelopmental Disorder; Organoids; Phase; Phenotype; Physiology; Poison; Research; Research Project Grants; Research Training; Role; Science; System; Technology; Time; Training; Transcript; Untranslated RNA; Vision; Work; Zebrafish; base; behavioral phenotyping; blind; career; comparative genomics; cost; design; epigenomics; gene function; genetic element; genome wide association study; genome-wide; high throughput screening; high throughput technology; human disease; improved; in vivo; insight; knockout gene; meetings; molecular phenotype; monolayer; mutant; neuropsychiatric disorder; new technology; novel; promoter; reverse genetics; scale up; single-cell RNA sequencing; skill acquisition; tool; training opportunity; vertebrate genome

PROJECT SUMMARY It has been almost two decades since the completion of the Human Genome Project, yet much of the genome remains poorly understood. In particular, the function of most of the noncoding genome, which makes up almost 98% of the human genome, is unknown. To be sure, these noncoding sequences do carry functional relevance, as multiple large-scale biochemical studies, genome-wide association studies, and comparative genomics have suggested. However, direct evidence linking a noncoding genomic region to its function is limited. The development of CRISPR-Cas9 has, for the first time, made functional interrogation of the noncoding genome accessible. Indeed, high-throughput perturbation studies in mammalian cells have been instrumental in identifying the functions of many noncoding genomic regions. However, in vitro cell cultures are phenotypically limited to observations at the level of single cells, monolayers, or organoids. These systems cannot fully recapitulate the wide array of animal physiologies and behaviors relevant to human health. Whole animals, on the other hand, model a plethora of complex biological processes such as embryonic development, cancer and metastasis, infection and immunity, neurodevelopmental and neuropsychiatric disorders and many more. Scaling up CRISPR-Cas9 technology for in vivo functional screens, however, has been extremely challenging due to the cost, labor, and time required to generate mutant animals. A platform that enables large-scale CRISPR-based perturbation in vivo will open new avenues for whole animal functional genetic screens. I have developed Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform that makes large-scale reverse- genetic screens possible in zebrafish. The platform is robust and can be scaled-up to target thousands of genomic regions. This proposal outlines a five-year plan to use the platform to systematically interrogate the functions of the noncoding genome in vivo. I plan to use comparative genomics to identify two classes of highly conserved but poorly understood noncoding genomic regions: (1) Poison Exons and (2) Enhancers. Subsequently, I plan to systematically perturb these elements and assess their functional roles in regulating vertebrate development and behavior. These exploratory aims will be aided by the simultaneous development of new and improved tools for genetic perturbation and phenotyping. The tools I develop and the knowledge I gain from the proposed research project will allow me to establish a successful independent career in functional genetics. This project also offers significant growth and training opportunities in the form of both formal courses and meetings as well as collaborations. I have assembled a team of mentors, collaborators, and advisors— consisting of experts in Comparative Genomics, Bioinformatics, and Developmental Biology—whose support and mentorship will provide me the necessary training needed for successful completion of the proposed aims. My research training will be complemented by additional trainings in professional skill development such as mentoring, lab management, and budgeting ensuring a successful transition to an independent research career.

项目概要 人类基因组计划完成已近二十年，但基因组的大部分内容 仍然知之甚少。特别是大部分非编码基因组的功能，它们几乎构成了 人类基因组的98%是未知的。可以肯定的是，这些非编码序列确实具有功能相关性， 随着多项大规模生化研究、全基因组关联研究和比较基因组学的进展 建议。然而，将非编码基因组区域与其功能联系起来的直接证据是有限的。这 CRISPR-Cas9的开发首次对非编码基因组进行了功能询问 可以访问。事实上，哺乳动物细胞的高通量扰动研究在以下方面发挥了重要作用： 鉴定许多非编码基因组区域的功能。然而，体外细胞培养物的表型 仅限于单细胞、单层或类器官水平的观察。这些系统不能完全 概括了与人类健康相关的各种动物生理学和行为。整个动物，在 另一方面，模拟大量复杂的生物过程，例如胚胎发育、癌症和 转移、感染和免疫、神经发育和神经精神疾病等等。 然而，扩大 CRISPR-Cas9 技术用于体内功能筛选一直极具挑战性 由于产生突变动物所需的成本、劳动力和时间。一个可以实现大规模的平台 基于 CRISPR 的体内扰动将为整个动物功能遗传筛选开辟新途径。我有 开发了多重混合 CRISPR 液滴 (MIC-Drop)，这是一个可以进行大规模反向 斑马鱼的遗传筛选成为可能。该平台非常强大，可以扩展到数千个目标 基因组区域。该提案概述了一个五年计划，旨在利用该平台系统地询问 体内非编码基因组的功能。我计划使用比较基因组学来识别两类高度 保守但知之甚少的非编码基因组区域：(1) 毒外显子和 (2) 增强子。 随后，我计划系统地扰动这些元素并评估它们在调节中的功能作用 脊椎动物的发育和行为。这些探索性目标将得到同步发展的帮助 用于遗传扰动和表型分析的新的和改进的工具。我开发的工具和我学到的知识 从拟议的研究项目中获得的收益将使我能够在功能领域建立成功的独立职业生涯 遗传学。该项目还以正式课程的形式提供重要的成长和培训机会 以及会议和合作。我组建了一个由导师、合作者和顾问组成的团队—— 由比较基因组学、生物信息学和发育生物学专家组成——他们的支持 指导将为我提供成功完成拟议目标所需的必要培训。 我的研究培训将得到专业技能发展方面的额外培训的补充，例如 指导、实验室管理和预算确保成功过渡到独立研究职业。