Author Archives: Claire Engstrom

Throughout my eight weeks as a BSURFer, we had various faculty members present their research along with their inspiring stories about their journey to a faculty postition. An overall theme in each of their stories was the uncertainty about their path in life. Most of the professors had never thought about entering academia and a few had gone to medical school only to fall in love with research later. Of these faculty seminars, my favorite was Nutrition: the Human Microbiome (and me) by Dr. Lawrence David.

Dr. David’s research focuses on how dietary compounds stimulate growth and metabolism of gut microbes. Previous experiments explored the differences between high-fiber plant-based diets and low-carb animal-based diets through the analyzing of stool samples. Currently, his lab is investigating the most beneficial pre-biotic supplements to feed the microbes in a human gut. Since a large part of staying healthy and preventing disease in a majority of the population is based on diet, the experiments conducted in the David Lab are important for the overall health of the masses.

While his research was amazing to learn about, the advice portion of his presentation is what impacted me the most. He advised us to stay with our undergraduate lab for a long period of time, because good science can’t be done in just a semester. He also discussed his experience with graduate school because though he had some uncertainties at first, he realized that the experiments he was conducting made him happy and he enjoyed the process of scientific research. His final piece of advice was about knowing when to graduate. Once he finally felt like he knew what he was doing and became comfortable, his PI told him it was time to graduate. When he felt comfortable in his understanding of research, he stopped learning new things and stopped growing as a person, so it was time to take on a new adventure and learn something different. This constant sacrifice of intellectual comfort for new experiences is the difference between good and great, so I’ll be sure to carry that piece of advice with me through the rest of my journey in science.

A majority of Duchenne’s muscular dystrophy cases are caused by a frameshift mutation in the DMD gene and the deletion of exon 51 with CRISPR-Cas9 can restore the reading frame in many patients. However, there are thousands of gRNA sequences to choose from with a deletion frequency of only 2%, so which gRNAs in introns 50 and 51 are the most efficient? To test this, I designed 10 gRNAs in both introns and transfected 293T cells with these gRNAs and a CjCas9 plasmid. I harvested these cells, ran a PCR with designed surveyor primers and ran these fragments on a gel. No new bands were seen that were different from my no-gRNA control lane so the gRNAs I designed did not make an indel. I will test 2 new gRNA sequences with the same method and expect to see two bands unique to the gRNA-transfected cells. The results from my experiment will be positive controls for my mentor’s project of developing a high-throughput screening method of hDMD-targeted gRNAs. With broader and quicker gRNA screening, the CRISPR-Cas9 tool will be more efficient and used as a human medical treatment option for patients with DMD.

Dr. Charles Gersbach began his training as a biomedical engineer while he was an undergraduate at the Georgia Institute of Technology. As the son and nephew of many engineers, he decided to major in chemical engineering and was never exposed to biomedical research. After watching his friends enter the workforce at companies like Doritos, he realized that those jobs never interested him and he searched for other career options. During his last summer as an undergraduate, Dr. Gersbach worked in a lab and fell in love with research. Due to his lack of experience, he had trouble applying for graduate programs, but was admitted to Georgia Tech and got a PhD in Biomedical Engineering.

Now, Dr. Gersbach is a professor of Biomedical Engineering at Duke where he teaches two classes: one is a requirement for BME undergraduates and the other is a BioDesign elective course. He enjoys teaching the design course because the students are much more engaged. Since it is an elective and the course material includes innovative, cutting-edge methods, the students have a desire to learn which makes teaching it much more enjoyable. His favorite part about teaching though is watching his students go off to do amazing things in science. Watching graduate students that he mentored in his lab earn faculty positions at other universities and publish papers gives him the satisfaction of knowing that he taught them well. Nurturing young students to become contributors in his field is the best part of teaching for him.

Though Dr. Gersbach is well-established in his field, he has no lack of embarrassing stories within the lab. Due to his busy schedule, he has less time to spend in the lab doing actual experiments but is sometimes asked to help when his students go out of town. During one Christmas season, a few of his graduate students asked him to passage their cells while they went home for a few days. He forgot and the students returned to find all of their cells overconfluent and dead. They asked him to help the next year as well, but he accidentally contaminated the cells and again they died. Since then, they’ve stopped asking him to passage their cells and found other arrangements.

When asked about any advice he has for young undergraduates looking to get started in research, Dr. Gersbach told me to take advantage of the many opportunities offered at Duke. Since he was late in entering research, it took him nearly ten years to catch up, so programs such as BSURF are important to take advantage of. He also advises to try many different things and explore the various topics within science. He never thought he would like research until he worked in a lab, so sampling diverse subjects can help young students to learn about the careers they may want in the future. Overall, I enjoyed interviewing Dr. Gersbach and learning about the background of my mentor. It taught me there is always time to change directions as I discover my interests and to take advantage of the multitude of opportunities that Duke has to offer.

The Gersbach lab focuses on developing innovative methods in molecular and genetic engineering for applications in regenerative medicine, treating genetic disease, and enhancing understanding of fundamental biological processes. More specifically, many members of the lab aim to treat Duchenne’s muscular dystrophy with CRISPR-Cas9 genetic engineering tools by creating an insertion or deletion mutation that restores the reading frame of the DMD gene.

So far, their methods have been successful in skipping an exon to allow the production of dystrophin, though the deletion efficiency has been close to 2%. Though this is satisfactory for restoring muscle strength in mice, 2% is too low for humans and the methods would not be applicable to other genetic diseases. This inefficiency may be caused by the choice in guide RNAs that lead the Cas9 to the desired deletion site, as some gRNAs have been shown to have higher mutation rates than other. However, there are thousands of possible gRNAs and gRNA combinations for each exon and testing each one is tedious and time-consuming. My mentor’s project is developing a high-throughput strategy for screening pairs of gRNAs for hDMD exon deletions. For my project this summer, I will be testing 5 gRNAs on each side of exon 51 to determine which is most efficient to use as a positive control for my mentor’s screening techniques. By targeting both sides, my designed Cas9 will delete the exon and restore the reading frame shift that was caused by a mutation in an earlier exon. I can then sequence the DNA to see how many of the cells I transfected have exon 51 deleted and how well each gRNA worked.

In the future, this will hopefully lead to more frequent deletions with the use of CRISPR and Cas9 so that these genetic engineering tools can be used to treat those born with genetic diseases.