CRISPR-KI & iPSC (2/2): From Skin Cells to New Hope

7 days ago
5 min read

Updated: 6 days ago

In our previous article, we discovered CRISPR, that revolutionary tool capable of editing DNA with surgical precision. Today, we'll see what happens when we combine CRISPR with another scientific revolution: iPSC cells.

This combination opens fascinating possibilities for studying genetic diseases, even the rarest ones. But like any cutting-edge technique, it has its promises and challenges.

🔄 Quick Reminder: iPSCs

Induced pluripotent stem cells (iPSCs) are those "magic cells" we discussed in our "Understanding Science" series. In summary: we can take an ordinary skin cell, reprogram it, and transform it into any type of human cell—neurons, heart cells, liver cells...

It's like having a magic wand that transforms a mundane cell into a specialized cell on command.

🤝 The CRISPR + iPSC Marriage: Two Possible Approaches

Now imagine combining these two revolutions. On one side, CRISPR can modify DNA with precision. On the other, iPSCs can become any cell type. There are two ways to marry them depending on the situation:

🏥 Classic Approach: Patient First

When a biocollection exists, here's the usual process:

Collect cells from patients (skin, blood)
Reprogram them into iPSCs
Differentiate them into the affected cell type
Compare them with cells from healthy individuals

🧬 Alternative Approach: CRISPR First

When there's no biocollection yet, we can reverse the process:

Start with healthy cells reprogrammed into iPSCs
Use CRISPR to insert disease mutations
Transform these cells into the type affected by the disease
Compare cells with/without mutations

It's like creating perfect twins in the laboratory, where one would have the disease and the other wouldn't!

CRISPR-KI & iPSC for modelling LMBRD2 gene mutation

🔬 "Knock-In": The Art of Adding with Precision

The term "Knock-In" (KI) deserves attention. Unlike "Knock-Out" which removes a gene, "Knock-In" adds or replaces a precise DNA sequence.

✏️ The Text Editor Analogy

Imagine you're correcting a book:

Knock-Out = deleting an entire paragraph
Knock-In = replacing one specific word with another

To study genetic diseases, we often need to insert the exact mutation that causes problems. That's where Knock-In becomes indispensable.

🎯 Knock-In Advantages

🟢 Surgical precision: We insert exactly the desired mutation

🟢 Perfect control: We know exactly what we've changed

🟢 Reproducibility: Same modification in all laboratories

🟢 Flexibility: We can test different mutations one by one

🧪 The Process: From Cell to Disease Model

Let's see concretely how this technique works, step by step:

📋 Phase 1: Obtaining Cells

Everything starts with a simple sample: a few skin cells or a blood draw. These cells are then reprogrammed into iPSCs in the laboratory, regaining their ability to become any cell type.

✂️ Phase 2: Genetic Editing

This is the key moment! Scientists introduce CRISPR into the iPSCs. The tool searches for the exact location to insert the mutation, makes its precise cut, and the cell repairs by inserting the new sequence.

The challenge: Not all cells cooperate. It often takes many attempts.

🧬 Phase 3: Selection and Verification

Researchers must now identify which cells have received the modification. It's genetic detective work, where each cell is analyzed to verify that:

The mutation was properly inserted in the right place
There are no errors elsewhere in the DNA
The cells remain healthy

🧠 Phase 4: Differentiation

Now comes the magical transformation. The modified iPSCs are "convinced" to become the cell type affected by the studied disease. For a neurological disease, they become neurons. For heart disease, they become heart cells.

This step requires much patience and expertise.

🔍 What Researchers Can Discover

Once they have their cellular models—cells identical except for the studied mutation—scientists can finally answer fundamental questions:

🧬 Basic Biological Questions

🔬 Where does the mutated protein go? Does it localize to the right place in the cell?🔬 How much protein is produced? Is there more or less than with the normal gene?🔬 Does the protein function? Does it retain its normal capabilities?

🧠 Questions About Cellular Function

🔬 Do cells develop normally? Do they grow and divide correctly?🔬 Which other proteins are affected? Does the mutation have cascade effects?🔬 Do cells survive? Do they die more easily than normal cells?

💊 Therapeutic Questions

🔬 Can we correct the problem? Do certain medications improve the situation?🔬 How do cells react to treatments? What are the beneficial or toxic effects?

⚖️ Strengths and Weaknesses of the Approach

✅ Undeniable Assets

🟢 Availability: Allows starting even without an established biocollection

🟢 Perfect control: Identical cells except for the studied mutation

🟢 Reproducibility: Same material in all laboratories worldwide

🟢 Ethics: No ethical problems related to embryonic cells

🟢 Flexibility: Possibility to test different mutations

⚠️ Limitations to Know

🔴 Distance from reality: Cells in culture are not a human being

🔴 Artificial genetic background: These cells don't have the genetic history of real patients

🔴 Technical complexity: Difficult, expensive, sometimes temperamental technique

🔴 Validation necessary: Results must be confirmed on real patient cells

💰 Practical Realities

This cutting-edge research requires considerable resources:

💸 Financial Investment

🔹 Specialized equipment: Sterile cell culture laboratory

🔹 Expensive reagents: CRISPR tools, special culture media

🔹 Expert personnel: Technicians trained in advanced techniques

🔹 Analyses: Sequencing, functional tests

⏰ Variable Duration

Timelines depend on many factors: mutation complexity, target cell type, CRISPR efficiency, differentiation quality... Some projects succeed quickly, others take much longer.

🎯 Application to LMBRD2: Our Particular Case

Let's now address our specific situation with LMBRD2. Our case perfectly illustrates why the CRISPR-KI approach can be strategic.

🏥 Our Situation: Patients Yes, Biocollection No

We're fortunate to be in contact with several families affected by LMBRD2. However, organizing a formal biocollection—with all the necessary ethical and administrative authorizations—would take about 6 months.

🚧 The Time Dilemma

Facing this temporal constraint, our scientific partner, Dr. Alban Ziegler, proposed a two-phase strategy:

🎯 Phase 1: Start now with the CRISPR-KI iPSC approach

🎯 Phase 2: Validate and deepen with a real patient biocollection

🔬 Our CRISPR-KI Strategy

We would use this technique to create our own LMBRD2 neuronal models, focusing on recurrent mutations like Arg483His and Trp123Arg.

🔍 What We Hope to Discover

🔍 Protein localization: Where does LMBRD2 go in neurons? Does it stay there when mutated?

🔍 Gene expression: Do mutations change the amount of protein produced?

🔍 Neuronal development: Do neurons with LMBRD2 mutations develop normally?

🔍 Cellular functions: Which processes are disrupted by LMBRD2 dysfunctions?

🔄 Future Validation

This approach will give us valuable initial clues. When our biocollection is operational, we can compare our CRISPR-KI results with real LMBRD2 patient cells.

It will even be an excellent test: if our CRISPR-KI models give results consistent with patient cells, it will validate the approach!

💡 Conclusion: Pragmatism and Vision

The CRISPR-KI iPSC technique perfectly illustrates the evolution of modern research: rather than waiting for perfect conditions, we use available tools to move forward.

For ultra-rare diseases like LMBRD2, this approach can make the difference between years of waiting and immediate research startup. It's not perfect, but it's a concrete beginning.

And when we have access to real patient cells, we'll already have a solid foundation of knowledge and experience to go further, faster.

The important thing is to start.

Questions: contact@lmbrd2.org