In vivo functional screen of noncoding genetic elements

非编码遗传元件的体内功能筛选

基本信息

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
项目类别：
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

项目摘要

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特别是，大多数非编码基因组的功能仍然知之甚少。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发展然而，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。 CRISPR-Cas9的开发首次对非编码基因组进行了功能询问事实上，哺乳动物细胞的高通量扰动研究在这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，体外细胞培养物具有表型特征。仅限于单细胞、单层或类器官水平的观察，这些系统无法完全进行。概括了与人类健康相关的各种动物生理学和行为。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，将 CRISPR-Cas9 技术扩展到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，这是一个能够实现大规模生产的平台。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。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该平台功能强大，可以针对斑马鱼进行基因筛选。该提案概述了使用该平台系统地询问基因组区域的五年计划。我计划使用比较基因组学来识别两类高度的非编码基因组的功能。保守区域，但对非编码基因组知之甚少：（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该项目还以正式课程的形式提供重要的成长和培训机会。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——他们的支持导师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。