How scientists are using CRISPR/Cas9 to edit genes in stem cells, creating disease models and advancing future therapies
Our bodies are made of trillions of cells, each performing a specialized job. But at the very beginning of life, we start with pluripotent stem cells. Think of these as the master key cells; they have the potential to become any cell type in the body—a neuron in your brain, a cardiomyocyte in your heart, or a podocyte in your kidney.
Imagine a world where we could repair faulty genes before a child is even born, preventing devastating diseases from ever taking hold. While this sounds like science fiction, scientists are laying the groundwork for this future by mastering the art of genetic editing.
Sometimes, a person is born with a tiny "typo" in their genetic code, their DNA. One such typo occurs in a gene called NPHP1. When both copies of this gene (one from each parent) are broken, it leads to a severe childhood kidney disease called nephronophthisis, which often results in kidney failure. There is no cure.
To understand this disease and test potential therapies, scientists need a way to study it in a dish. The best way? Create it themselves. This article explores how researchers are using a revolutionary gene-editing tool, CRISPR/Cas9, to precisely generate these disease models by knocking out the NPHP1 gene in human pluripotent stem cells .
Master key cells with the potential to become any cell type in the body, providing the raw material for disease modeling and regenerative medicine.
Mutations in genes like NPHP1 can cause severe diseases. Creating precise models of these mutations helps researchers understand and develop treatments.
You've probably heard of CRISPR. It's often described as "molecular scissors," and that's a great starting point. The system has two key parts :
This is a protein that can cut the DNA double helix at a precise location.
This is a small piece of RNA that acts like a GPS, guiding the Cas9 scissors to the exact spot in the vast genome that needs to be cut.
The cell doesn't like having its DNA cut. It rushes to repair the break. Scientists can hijack this repair process to introduce specific changes. In this case, the goal was not to fix a gene, but to break it on purpose to create a disease model—a process known as "knockout."
Researchers design a guide RNA that matches the specific DNA sequence of the NPHP1 gene they want to edit.
The guide RNA binds to the Cas9 protein, forming the CRISPR/Cas9 complex that can search through the genome.
Once the complex finds the matching DNA sequence, Cas9 cuts both strands of the DNA double helix.
The cell attempts to repair the broken DNA, either through error-prone NHEJ or precise HDR, allowing researchers to introduce specific changes.
Let's dive into a specific experiment where scientists aimed to create a biallelic knockout of the NPHP1 gene—meaning they deactivated both copies of the gene in human pluripotent stem cells.
The challenge with a biallelic knockout is ensuring you successfully edit both copies of the gene. The researchers used a clever, practical two-part strategy :
They used CRISPR/Cas9 to make a cut in the NPHP1 gene. When the cell tried to repair it using a messy process called Non-Homologous End Joining (NHEJ), it often inserted or deleted a few DNA letters. This caused a "frameshift mutation," which is like changing the punctuation in a sentence—it scrambles the entire message that follows, rendering the gene useless. This successfully knocked out one copy (allele).
To knock out the second copy with 100% efficiency, they used a more sophisticated trick. Along with the CRISPR/Cas9 machinery, they provided the cell with a single-stranded DNA template (ssODN). This template was designed not to fix the gene, but to cause the cell to delete a large, critical chunk of it when it used the high-fidelity Homology-Directed Repair (HDR) pathway.
The beauty of this method is that by using two different repair outcomes (a small frameshift and a large deletion), scientists could easily distinguish and confirm that both gene copies had been successfully knocked out.
| Research Reagent | Function in the Experiment |
|---|---|
| Human Pluripotent Stem Cells (hPSCs) | The "raw material." These blank slate cells have the potential to become any cell type, including kidney cells, for disease modeling. |
| CRISPR/Cas9 Ribonucleoprotein (RNP) | The core editing machine. The Cas9 protein and guide RNA are pre-assembled for high efficiency and reduced off-target cutting. |
| Single-Stranded Oligodeoxynucleotide (ssODN) | The "trojan horse" repair template. This small DNA strand is designed to trick the cell's repair machinery into deleting a large part of the target gene. |
| Electroporator | The delivery system. This device uses a small electrical pulse to temporarily open pores in the stem cell membranes, allowing the CRISPR/RNP and ssODN to enter. |
The experiment was a resounding success. The researchers didn't just assume it worked; they proved it through rigorous testing:
This new stem cell line, now a perfect in vitro model of nephronophthisis, is a powerful new tool for research.
| Cell Sample | Allele 1 Status | Allele 2 Status | Resulting Genotype |
|---|---|---|---|
| Unedited (Wild-Type) | Normal NPHP1 | Normal NPHP1 | Healthy |
| Clone A1 | 4-bp Deletion (Frameshift) | 110-bp Deletion | Biallelic Knockout |
| Clone B3 | 1-bp Insertion (Frameshift) | 110-bp Deletion | Biallelic Knockout |
| Clone C5 | Normal NPHP1 | 110-bp Deletion | Heterozygous |
| Cell Type | NPHP1 Protein Detected? | Intensity (Relative to Control) |
|---|---|---|
| Control Kidney Cells | Yes | 100% |
| NPHP1-KO Kidney Cells | No | 0% |
Creating this specific NPHP1-knockout stem cell line is about far more than just breaking a gene. It's a foundational step forward. These customized cells provide a limitless source of human kidney cells that carry the exact genetic error found in patients. This allows scientists to:
Uncover why a broken NPHP1 gene leads to kidney failure at a cellular level.
Test thousands of potential drug compounds to find one that can rescue the sick kidney cells in a dish.
The same practical biallelic deletion strategy can be adapted to correct, rather than create, mutations.
By playing the role of cellular fixer-uppers—first learning to create precise breaks to model disease—scientists are mastering the tools they will one day need to perform flawless genetic repairs. This research brings us one step closer to future gene therapies for a wide range of genetic disorders.