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Principal Investigator: Aki Ushiki
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $114,015
Funding agency: National Human Genome Research Institute
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.
Terms: <ATAC sequencing><ATAC-seq><ATACseq><Address><Adeno-Associated Viruses><Assay><Assay for Transposase-Accessible Chromatin using sequencing><Associated Viruses><Bioassay><Biological Assay><Body Tissues><Brain><Brain Nervous System><CRISPR><CRISPR editing screen><CRISPR screen><CRISPR-based screen><CRISPR/Cas system><CRISPR/Cas9 screen><Catalogs><Cell Body><Cells><ChIP Sequencing><ChIP-seq><ChIPseq><Chromosome Mapping><Clustered Regularly Interspaced Short Palindromic Repeats><Code><Coding System><Color><Complex><Cultured Cells><DNA><DNA Sequence><DNA Transposable Elements><Data><Deoxyribonucleic Acid><Dependoparvovirus><Dependovirus><Development><Disease><Disorder><Double-Stranded DNA><Ectoderm><Electroporation><Elements><Embryo><Embryonic><Encephalon><Endoderm><Enhancer Elements><Enhancers><Ensure><Fallopian Tubes><Female><Fertilized Egg><Fertilized Ovum><Functional RNA><Future><GWA study><GWAS><Gene Localization><Gene Mapping><Gene Mapping Genetics><Genes><Genetic Alteration><Genetic Change><Genetic Enhancer Element><Genetic defect><Genetics-Mutagenesis><Genome><Genomics><Germ Layers><Glycoproteins><Goals><Guide RNA><Heart><Heterozygote><High Throughput Assay><Human Genome><In Vitro><Individual><Investigators><Knock-out><Knockout><Lead><Libraries><Linkage Mapping><Liver><Mammalian Oviducts><Measures><Mentors><Mesoderm><Methods><Mice><Mice Mammals><Murine><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutation><Non-Coding><Non-Coding RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Nucleic Acid Regulator Regions><Nucleic Acid Regulatory Sequences><Nucleotides><Organism><Pb element><Penetration><Phase><Phenotype><Publications><Regulatory Element><Regulatory Regions><Reporter><Reporting><Research><Research Personnel><Researchers><Salpinx><Satellite Viruses><Scientific Publication><Serotyping><Single Base Polymorphism><Single Nucleotide Polymorphism><Specificity><System><Techniques><Technology><Testing><Thick><Thickness><Time><Tissues><Total Human and Non-Human Gene Mapping><Training><Transgenic Mice><Transposable Elements><Transposase><Untranslated RNA><Uterine Tubes><Variant><Variation><Zona Pellucida><adeno associated virus group><assay for transposase accessible chromatin followed by sequencing><assay for transposase accessible chromatin seq><assay for transposase accessible chromatin sequencing><assay for transposase-accessible chromatin with sequencing><base editor><career><catalog><cell type><chromatin immunoprecipitation-sequencing><clustered regularly interspaced short palindromic repeats screen><developmental><ds-DNA><dsDNA><electroporative delivery><enhancer sequence><extracellular><functional genomics><gRNA><gain of function><gastrulation><gene electrotransfer><gene function><genetic enhancer sequence><genetic mapping><genetic regulatory element><genome mutation><genome scale><genome wide association><genome wide association scan><genome wide association studies><genome wide association study><genome-wide><genomewide><genomewide association scan><genomewide association studies><genomewide association study><genomic data><genomic data-set><genomic dataset><genomic tools><heavy metal Pb><heavy metal lead><hepatic body system><hepatic organ system><heterozygosity><high throughput screening><high throughput technology><human disease><human whole genome><in vivo><living system><loss of function><noncoding><novel><oviduct><pregnant><prevent><preventing><prime editor><promoter><promotor><screening><screenings><single nucleotide variant><tool><whole genome association analysis><whole genome association studies><whole genome association study><zygote>