Inducible Degrons Help Researchers Catalog Gene Function
Reversible degron tools make it possible to observe an immediate phenotype before it is complicated or compromised.
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A large portion of human protein-coding genes have not been well-studied and, among those that have been, only a subset have well-characterized functions. In 2022, The National Institutes of Health launched the Molecular Phenotypes of Null Alleles in Cells (MorPhiC) program to help better understand the function of every human gene.
MorPhiC aims to develop a catalog of molecular and cellular phenotypes for null alleles – a variation in a DNA sequence that results in no gene product – for every human gene, which will be made available for broad use by the research community. Dr. Mazhar Adli, the Thomas J. Watkins Memorial Professor of Tumor Genomics at Northwestern University Feinberg School of Medicine, is involved in Phase 1 of the project.
This phase is focused on optimizing available methods to create null alleles and measure their phenotypic effects in a targeted subset of 1,000 protein-coding genes.
Technology Networks caught up with Adli following his presentation at the Society for Laboratory Automation and Screening (SLAS) 2025 meeting to learn more about his research on the characterization of essential human genes and the benefits of using degrons to study gene function.
What is a degron?
A degron is a transferable degradation signal, such as a short amino acid sequence, which can be attached to a protein for inducible and targeted proteasomal destruction.
Characterizing tricky “essential genes”
“Humans have about 20,000 protein-coding genes and we have only characterized the function of about 5%. Our understanding is also biased towards a limited number of genes in a limited number of settings, mostly in cancer and some in neuroscience,” said Adli.
In some instances, this lack of understanding comes from inherent challenges in the methods used to characterize gene function, as is the case with “essential genes”. Adli explained, “For some genes, it’s impossible to even study their functions because, typically, we study genes by removing them from cells to observe certain phenotypes. However, we have a group of ‘essential genes’ that we cannot remove because it results in cell death.”
To overcome this, Adli and colleagues utilize reversible auxin-inducible degron (AID) tools that induce rapid depletion of target proteins in minutes. This makes it possible to observe an immediate phenotype before it is complicated or compromised by secondary effects and/or adaptation.
“Some genes are important after 48 hours; however, some are so important that within a few hours of their removal, we observe massive cell death. Using this technology, we are now able to characterize these essential genes,” Adli said.
What is the auxin-inducible degron system?
The auxin-inducible degron system is a tool that can conditionally induce the degradation of any protein through proteasomal degradation using the plant hormone auxin, which allows researchers to study protein function in living tissues. Normal human cells have all the necessary components except the auxin-inducible TIR1 protein.
Adli’s lab engineers human induced pluripotent stem cells (hiSPC) to express the TIR1. They then use CRISPR-Cas9 technology to add the auxin inducible degron (AID) tag to specific endogenous gene loci. Cells express the tagged protein normally until treated with auxin, which induces rapid, reversible protein degradation.
The need for a gene catalog
Understanding what human genes do is no easy feat; many genes have multiple functions and can behave differently depending on the type of cell in which they are expressed. Genes may also turn on and off depending on the cell’s environment, age and relation to surrounding cells.
“Historically, when investigating gene function, we would have a specific phenotype in mind. For example, if it's a cancer study, you may look at cell proliferation. However, when you don't know the function of a gene, then there are endless phenotypes to study, such as proliferation, cell death, cell signaling, responses to growth hormones, etc.,” stated Adli.
“In addition, we are still limited by the type of phenotypes that can be assayed. Therefore, I think it is important to have a catalog and a system that is openly available to other researchers. In my lab, we are generating these degron-tagged cell lines as a resource, and we are going to make them openly available to the research community so that other researchers can start using them and characterizing these genes for assays with their expertise. This will collectively let us accumulate a lot more data about the functional outcomes of these null alleles.”
To study as broad a range of cell types as possible, Adli and colleagues apply genome engineering and targeted protein depletion strategies to induced pluripotent stem cells (iPSCs). These cells offer multiple benefits for in vitro research such as the ability to differentiate into most somatic cell types.
“If you look at the number of publications per gene, 95% of genes don’t have any published research about their functions. But for five percent there is a lot of published information, for example, if you think about cancer cells the TP53, EGFR and KRAS genes are well studied. If we keep studying the same cancer cells, we will most likely keep finding similar genes, so this doesn’t help with removing the bias in our understanding,” said Adli.
“Using iPSCs allows us to study several developmental phenotypes. We can take these cells and differentiate them into multiple different cell types. This gives us access to a multi-cellular system that by knocking out a gene in one particular cell, we can differentiate into multiple other phenotypes and lineages.”
In addition, the iPSCs Adli’s team uses are derived from human cells providing a model that more accurately mimics what happens in human cells and tissues than traditional model organisms. “Although the model organisms are very powerful, none of them are human, and we need to understand the function of these genes in human relevant settings,” Adli explained.
Understanding the phenotypic effects of removing a gene's protein product could provide wide-ranging insights into their biological function. Adli hopes that such data will provide the research community with a foothold for understanding the mechanisms through which genes act to produce phenotypes.
Adli concluded, “At least 14–15,000 protein genes are active in a given cell, and how they all work together to make a particular cell type versus another cell type is still not fully understood. Once we have a catalog of gene functions and with the advances of computational biology, especially with AI, we will be able to group these genes to understand how they work together to establish a specific phenotype or cellular state. These efforts will pave the way for effective therapeutic intervention or engineering human cells for therapeutic purposes.”
