Saturday, April 20, 2024
4/20/2024
PROJECT SUMMARY Mutations in gene regulatory elements (REs) are a major cause of human disease. For example, the majority (>90%) of disease related genome wide association studies (GWAS) found associations with variants in non- coding and likely regulatory regions in the genome. Despite their importance, the code and grammar of these regulatory elements remains largely unknown making the understanding of how mutations in these sequences can lead to disease even more complex. Regulatory elements can be identified in a genome-wide manner using techniques such as ChIP-seq or ATAC-seq. However, these methods are descriptive and do not provide a functional readout that tests whether these elements are indeed functional. Massively parallel reporter assays (MPRAs) and CRISPR-based screens have recently been developed to functionally characterized these elements in a high-throughput manner. However, most of these techniques use cultured cells to measure activity. As such, the activity and function of these elements and their variants in an organism has not been tested. Due to this, complex phenotypes, such as spatial-temporal, and tissue/cell type specificity and interactions cannot be assessed for these elements. In this K99/R00 application, I will develop technologies that will allow to functionally characterize regulatory elements and variants in a high throughput manner in mice. One of the biggest barriers that prevent high-throughput assays in mice is the zona pellucida that surrounds one-cell stage embryos and prevents double-stranded DNA to be inserted. Recent reports and my own preliminary data show that by utilizing adeno associated virus serotype 6 (AAV6) as a delivery tool, DNA can integrate into one-cell stage embryos. I plan to use AAV6 along with the PiggyBac transposase system, that allows for genomic integration in all three-germ layers, to develop MPRA in mice (Aim K1). To validate the effect of single nucleotide variants, I will develop large-scale CRISPR saturation mutagenesis assays in mice. This will be done by utilizing in vitro electroporation into embryos and base-editor or prime-editor transgenic mice (Aim K2). Finally, I will apply these technologies to generate a catalog of functional regulatory elements, including transposable elements, involved in differentiation of the three primary germ layers (Aim R1). The results from this proposal will provide novel in vivo high-throughput technologies that will enable to study regulatory elements and disease-associated variants at any developmental time stage in mice. My career goal is to lead an independent research group developing novel functional genomics tools in mice and studying the function of gene regulatory elements and their variants in tissue development and disease utilizing these technologies. To achieve this goal, I will receive experimental and computational training from my mentors Drs. Nadav Ahituv and Jay Shendure. This rigorous mentored support and results obtained in the K99 phase will ensure my transition to an independent investigator and future successful independent career.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
