In vivo functional screen of noncoding genetic elements

项目总结 人类基因组计划已经完成了近20年，但大部分基因组 人们对此仍然知之甚少。尤其是大多数非编码基因组的功能，它几乎构成了 98%的人类基因组是未知的。可以肯定的是，这些非编码序列确实带有功能相关性， 作为多项大规模的生化研究，全基因组关联研究和比较基因组学 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。这个 CRISPR-Cas9的开发首次对非编码基因组进行了功能查询 无障碍。事实上，哺乳动物细胞中的高通量扰动研究已经在 鉴定许多非编码基因组区域的功能。然而，在体外细胞培养是表型 仅限于在单细胞、单分子层或有机体水平上的观察。这些系统不能完全 重述与人类健康相关的动物生理和行为的广泛范围。整个动物，上 另一方面，模拟了过多的复杂生物过程，如胚胎发育、癌症和 转移、感染和免疫、神经发育和神经精神障碍等等。 然而，扩大CRISPR-Cas9技术用于活体功能屏幕一直是极具挑战性的 由于产生突变动物所需的成本、劳动力和时间。一个能够实现大规模 基于CRISPR的体内扰动将为整个动物的功能遗传筛选开辟新的途径。我有过 开发了多路混合CRISPR液滴(MIC-Drop)，这是一个使大规模反转的平台 斑马鱼的基因筛选是可能的。该平台非常强大，可以扩展到面向数千名 基因组区域。该建议概述了一个五年计划，利用该平台系统地审讯 体内非编码基因组的功能。我计划使用比较基因组学来鉴定两类高度 保守但知之甚少的非编码基因组区域：(1)有毒外显子和(2)增强子。 随后，我计划系统地干扰这些元素，并评估它们在调控中的功能作用 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鉴定的新的和改进的工具。我开发的工具和我所掌握的知识 从拟议的研究项目中获得的收益将使我能够在功能部门建立成功的独立职业生涯 遗传学。该项目还以两种正式课程的形式提供显著的增长和培训机会 以及会议和协作。我已经组建了一个由导师、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 而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职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的研究生涯。

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.