Molecular and cellular characterization of essential human genes.

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 targe 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-in 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 subviable
phenotypes in the International Mouse Phenotyping Consortium (IMPC). We propose to catalog
the cellular phenotypes (survival, proliferation, mitotic function, and differentiation) and molecular
phonotypes, 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.

Grant Number: 5UM1HG012649-04
NIH Institute/Center: NIH
Principal Investigator: Mazhar Adli