Title: Characterizing human genes by generating barcoded and inducible null alleles Abstract We propose to generate barcoded and conditional null alleles in a cellular system that can model early human development and a broad range of human diseases. We will establish a data production research and development center in response to the RFA-HG-21-029: Molecular Phenotypes of Null Alleles in Cells (MorPhiC) Phase 1, which aims to establish a catalog of molecular and cellular phenotypes of null alleles for ultimately every human gene, using in vitro multicellular systems. Our center will utilize a chemically inducible and reversible system that enables the rapid depletion of target proteins. The approach permits temporal control of protein levels to study the consequences of null alleles. We will utilize a super sensitive degron that rapidly degrades the target protein of interest in response to a low dose of auxin, a cell membrane diffusible small chemical plant hormone. We will combine CRISPR-based targeted locus engineering to homozygous knock-in a mini auxin-inducible degron (mAID) at the end of the target gene to create a chemically controllable switch to create null-alleles in an open-access human induced pluripotent stem (hiPSC) cell, which can be differentiated into various cell lineages and multicellular organoids to model human development and diseases. Notably, each AID-degron will also contain gene-specific barcodes, allowing tracking the fate of hundreds of thousands of null alleles when these engineered null alleles are pooled. The proposed approach is generalizable and can rapidly deplete target proteins codded by various classes of human genes. Our strategy will be particularly advantageous and critical to study the null phenotypes of essential genes, which cannot be studied by chronic depletion using genetic approaches (such as CRISPR KO) because the knock-out results in cell death. Therefore, to highlight the utility of our strategy, we prioritize creating null alleles by CRISPR mediated knock-in process to introduce barcoded AID degron in 250 essential genes. We chose genes implicated in human diseases and sub-viable phenotypes in the International Mouse Phenotyping Consortium (IMPC). We propose to catalog the cellular phenotypes (survival, proliferation, mitotic function, and differentiation) and molecular phenotypes, including gene expression and chromatin accessibility for select null alleles. This information will provide unique insights into the biological function of these developmentally critical genes. It will highlight the utility of establishing the chemically inducible degron system as a generalizable strategy for the goals of the MorPhiC consortium. The created barcoded and conditional null allele resource will provide a unique opportunity to temporally control the timing of null alleles in pluripotent stem state and various terminally differentiated cell types or multicellular organoid systems that can be generated from the pluripotent stem cells.
|Effective start/end date||9/22/22 → 6/30/27|
- National Human Genome Research Institute (1UM1HG012649-01)
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