A New Way to Listen: Gene-Edited Cells Reveal the Individual “Voices” of ENT1 and ENT2
Dr. Nayiar Shahid - 27 November 2025
Every cell in the human body needs a steady supply of nucleosides, small molecules used to build DNA and RNA. But many fast growing cells cannot make enough nucleosides on their own. Instead, they rely on special “gateways” in the cell membrane called equilibrative nucleoside transporters, or ENTs. ENT1 and ENT2, do most of the work.
For years, I kept returning to one question: How can we understand what ENT1 and ENT2 each do if they are always active at the same time? Yet no matter how many experiments we tried, ENT1 always overshadowed ENT2. It felt like trying to listen to two people talking at once, without ever being able to separate their voices.
This challenge became the motivation behind our study in the Hammond lab at the University of Alberta. If we could turn off ENT1, ENT2, or both, cleanly and precisely, we may finally see what each transporter does on its own. That knowledge could help us understand how drugs enter cells, how cancer and viral treatments work, and how cells cope when one or both of their major supply routes is removed.
Using CRISPR gene editing, we created exactly what we needed: a set of human kidney cell lines where ENT1, ENT2, or both were removed. These models, ENT1-KO, ENT2-KO, and the double knockout (E1E2-KO) allowed us to study one transporter at a time.
You have probably heard about CRISPR. It’s the gene editing machine. Well, let me tell you something. Gene editing is delicate work. It requires accuracy, persistence, and patience. After CRISPR makes its cut, just like a molecular surgeon (cutting out genes while leaving the rest of the cells intact), each edited cell must be isolated and grown from a single cell into a full clone, a process that takes several months.
Only then can we test the clones to find the ones where the gene is completely removed. Once we identified those clones, we performed the essential checks: ENT-specific RNA and protein were gone, transport activity was eliminated, and ENT1-deficient cells no longer bound NBMPR, a molecule that attaches only to ENT1. These checks assured us that each knockout cell line was exactly as intended. With these clean cell models, we could finally study ENT1 and ENT2 separately.
We found that ENT1 is the major transporter for many nucleosides, while ENT2 becomes especially important when cells are stressed or when ENT1 is missing. In the double-knockout cells, nucleoside transport dropped almost to zero, showing that ENT1 and ENT2 work together to handle nearly all nucleoside movement in cells.
The biggest surprises, however, were inside the cells. When one or both transporters were removed, the genes involved in purine metabolism, the recycling pathway that helps rebuild DNA and RNA, became more active, as if the cells were trying to make up for the lost major gateways. Growth patterns shifted too: cells missing ENT1 or ENT2 alone grew faster, while the double-knockout cells grew significantly slower, revealing how each transporter supports normal cell growth and energy balance in different ways.
One of the most important findings came from testing nucleoside-analogue chemotherapeutic drugs. Using our new cell models, we could clearly see how each drug behaved when ENT1, ENT2, or both were missing, giving us a better picture of how ENT1 and ENT2 influence drug action and effectiveness. Our results showed that different drugs depend on ENT1 and ENT2 to different degrees and surprisingly, ENT2 played a bigger role than expected in helping cells survive after treatment with several nucleoside-analogue chemotherapies. This insight is one of the strongest outcomes of using these new knockout models.
For me, this project began with one simple goal: to finally hear the individual “voices” of ENT1 and ENT2. Now, with these CRISPR-edited models, we can; and that clarity opens the door to better science and better medicines. Although our findings are preliminary, we now have a path toward more detailed studies that can explain how these drugs behave inside cells.
By pinpointing the distinct roles of ENT1 and ENT2, our work gives researchers a new tool to look at those secondary questions regarding drug transport and therapy development. These new cell lines may not seem to be significant in the grand scheme of human health, but we know the value in enabling scientists to test drugs more accurately and understand how nucleoside movement shapes cell health, improving future drug screening and deepening our knowledge of the cell’s most important molecular gateways. What we can be sure of is that as these novel advancements are recorded in history, our voice will be there forever.
Dr. Shahid is currently a member of the Basic Sciences Administrative Team and can be reached at nayiar@ualberta.ca.