In vivo functional screen of noncoding genetic elements

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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.