DREAMing about C2C12 cells under a microscope

Author: Ian Popp | Major: Chemical Engineering, Finance | Semester: Spring 2024

I met Dr. Nelson through my GNEG research class, and I chose to work with him alongside another student because we thought gene editing could be fun. Mary Jia, an extraordinary undergraduate researcher, and Allie Ivey, a doctoral student, would train us in CRISPR gene editing. In large part due to Dr. Nelson, I had recommendations that carried me to the Ole Miss Nanoengineering Research Experience for Undergraduates, a major win. I was grateful for Dr. Nelson, so I wanted to work more in the Nelson lab, knowing that Dr. Nelson had looked out for me and would continue to do so.

CRISPR-DREAM is a new gene editing tool developed by Dr. Nelson’s friend at Rice University. Using the power of friendship, he was able to get access to the tool, and he offered me a project to apply CRISPR-DREAM to Duchenne Muscular Dystrophy. Since I was given a project, I asked Dr. Nelson to be my mentor to write the research grant application. Although I had exposure to the cell culture part of the lab, gene editing is new, and I’ve found that CRISPR functions a lot like using a car.

Duchenne Muscular Dystrophy is a severe genetic disorder, impacting 1 in 3,500 male births worldwide. Duchenne Muscular Dystrophy is caused by mutations in the X chromosome for the gene that codes dystrophin, a critical protein for muscle cells’ cytoskeletons. Because the dystrophin is broken, muscle cells can’t function, causing a tragic early death.

After going through a literature review with Dr. Nelson, we found papers that suggest introducing utrophin can compensate for the dystrophin shortage. Using a mouse genome browser, we found possible sites for us to park our CRISPR-DREAM on the DNA that could upregulate utrophin. Now that we found the open parking spots, we had to build the vehicle with a respective guide which would take our CRISPR-DREAM complex to park in each respective spot. After designing the utrophin guides, Dr. Nelson decided that we should design other guides too that we would use after I finished my utrophin project, designing guides for four other genes that could be used in synergy with my utrophin gene guides. After getting carried away, we came up with over forty targets, and Dr. Nelson noted that we couldn’t have gotten so many guides without the Honors college research grant!

To continue the CRISPR-DREAM car analogy, we had to begin cloning the guides into the plasmids, like giving the directions for the driver where to go. There are three major steps in the cloning process. be Like typing out the street address in my phone for where we want to go, I phosphorylated the ends of each of the guide RNAs, creating an insert that could fit into the plasmid. In the ligation step, I cut open the backbone plasmid which allows the guide RNAs to be inserted into the incision, like texting the address for the driver’s GPS with poor cell service. After the ligation step, the complete plasmid, or driver who knows the directions, is inserted into the bacteria, or he starts the car. The bacteria then go onto petri dishes with the specific antibiotic, and the driver is tested to see if he knows the directions. The surviving colonies of plasmids, or drivers, should have the correct guide inserts, or directions. After extracting the plasmid from the bacteria, I can confirm that the plasmid knows the right directions by sending the DNA off to a corporate lab for sanger sequencing, like sending the driver to the DMV for a driving test. Just like communicating directions in a car, cloning usually requires many tries.

Because the procedure for cloning won’t always work, it is best to clone many guides at once. Thus, Dr. Nelson decided that we would be cloning my utrophin guides and the other targets he wanted to use. I had a nightmare as part of the DREAM team; I now had to clone forty guides instead of ten, and my future project was suddenly included in my current project. Luckily, Dr. Nelson informed me that I would be working alongside a graduate student from another lab as he made a few other guides for a CRISPR DREAM edit for his project, so we would be working together, forging a true DREAM team. Because his project differed from mine, I got a better idea for the process for selecting gene targets. Despite having over forty guides, I have completed the cloning procedures for the guides about six times, and I only have a few left. Although Dr. Nelson offered to let me save a few targets for later, I declined knowing that I couldn’t quit before I win big by finding the best guides!

I have begun cell culture experiments with mouse muscle cells. We can mix the plasmid into the delivery tool, like putting drivers in cars, creating a functional CRISPR DREAM solution to send to the cells. When CRISPR-DREAM arrives in its parking spot, it can begin working to make the cell change how much it makes of the target protein. After sending the editing tool to the cells, we will be able to evaluate protein expression from our target genes through Western blotting, which literally paints a picture of how much of our target protein is there in relation to other proteins. This will determine whether our gene target and edit are successful.

I am grateful for the experience that the Honors college research grant has created for me. Dr. Nelson has worked with me, spending hours each week with me. He has provided me with constructive feedback while also encouraging me to be independent in the lab. With this work, I intend to submit an abstract to BMES to present my research at a national conference. By attending a conference, I hope to network with other professionals to gain an edge for the future. My final goal with my grant is to publish this research.