Theo Roth Engineered a CRISPR Breakthrough
Theo Roth was sure he wanted to be an engineer. Despite growing up the son of two academics, he didn’t think he would be a researcher. He liked math, and he wanted to build things. Theo stuck to his plan to pursue engineering until he graduated from high school. Looking to get his feet wet in the working world before college, he found an internship at the NIH and moved to Bethesda to do research for the summer.
In the lab, Theo became fascinated with the experimental process. He loved the sense of freedom it provided: the freedom to ask his own questions, to determine how to answer those questions, to see something that had never been seen before. He also found parallels between engineering and biology. Biology, just like engineering, involved deciphering patterns and simplifying complex systems. Soon, Theo couldn’t imagine himself doing anything other than designing experiments.
On Early Mentorship
Theo spent his four undergraduate years at Stanford working in Dr. Matthew Scott’s lab, and he made the trip back to the NIH each summer to continue the research that he started in Dr. Dorian McGavern’s lab. In the McGavern Lab, Theo investigated immune responses in the brain using intravital microscopy, a technique that allows scientists to label and visualize cells in live mice. Theo recalls learning this imaging technique—playing around with different conditions, experimenting with new instruments, and watching the cells in real-time—as “the coolest thing ever.”
Theo remembers that the two of them got excited about the same questions: How do you approach a scientific question? What techniques do you need to carry out an experiment? How do you plan it, set it up?
The McGavern Lab was instrumental to Theo’s career not only because it provided his first window into biological research, but also because Dr. McGavern cultivated Theo’s scientific curiosity and growth. Theo remembers that the two of them got excited about the same questions: How do you approach a scientific question? What techniques do you need to carry out an experiment? How do you plan it, set it up? Theo also appreciated that his advisor was heavily engaged in the day-to-day operations of the lab. For example, if a piece of lab equipment broke, Dr. McGavern would often fix it himself. To Theo, it was clear that his mentor understood every part of the lab, from its physical infrastructure to its most advanced scientific findings, and how those parts fit together.
During Theo’s early days in the lab, Dr. McGavern would regularly sit down with him one-on-one to sift through data and results, looking through images or watching videos of labeled cells on loop. He would ask Theo to hypothesize why the cells might be moving around in a certain way, what his observations might tell him about the biology. “It didn’t matter [to Dr. McGavern] that I had very little scientific understanding of what was going on,” Theo recalls. Over time, Dr. McGavern taught Theo how to begin to make sense of complex biological systems, and his mentorship instilled both confidence and tenacity in Theo. Under Dr. McGavern’s guidance, Theo even gathered enough compelling data to publish a first-author Nature paper about the brain’s immune response to trauma during his senior year of college.
Driving a CRISPR Discovery
That wouldn’t be the last time Theo published a news-worthy Nature paper. Theo’s undergraduate experiences inspired him to pursue a dual MD/Ph.D. degree at the University of California, San Francisco, and to carry out his Ph.D. work under Dr. Alex Marson. Shortly before Theo joined, the Marson Lab published research that revealed a novel method for carrying out targeted CRISPR/Cas9 knockouts in T cells. That paper introduced another possibility: Could scientists could take advantage of this method in T cells to introduce new genes at specific genomic locations? If it were possible, using CRISPR to perform knock ins of long transgenes would be easier, faster, and cheaper than existing techniques that relied on viral gene delivery.
Theo set out to see if it could be done, taking advantage of his knack for experimental design. Developing a new method would require both a clever and an efficient discovery pipeline, one that would allow Theo and his collaborators to test various permutations in high-throughput. This would minimize the amount of uninformative trial-and-error and, instead, leverage the data from each experiment, using it to inform the next. Theo and his collaborators worked for months to create a pipeline that could analyze 96-well plates and automate a handful of experimental steps. Theo spent many late nights in lab, but he calls those nights “late nights spent intelligently.” Once the pipeline was up-and-running, it allowed Theo to run experiments and to optimize conditions very quickly.
Theo tested dozens of plates and thousands of electroporation conditions, ultimately developing a reliable method for transgene delivery using CRISPR. Theo even showed that this method could be used to replace a T cell’s endogenous antigen receptor, the TCR, giving the cell a new target. His result surprised scientists because the large, double-stranded templates that he transfected were previously thought to be toxic to cells.
When the paper outlining the new method was accepted, Theo and his collaborators celebrated their hard work and late nights with pizza and beer. But Theo didn’t waste any time. He was already planning his next round of experiments. “Technique development is only as exciting as the types of experiments it enables. I didn’t join a lab to do technical development, but to modify T cells to cure complex diseases.”
“Technique development is only as exciting as the types of experiments it enables. I didn’t join a lab to do technical development, but to modify T cells to cure complex diseases.”
Theo plans to focus the remainder of his MD/Ph.D. training on understanding disease processes so that he can later develop cell therapy products. He expects CRISPR/Cas9 to have powerful therapeutic applications in T cells, which he characterized as “magical cellular machines.” Genetically-modified T cells, he believes, could provide a form of super-personalized medicine, where scientists would engineer specific genetic changes to treat individual patients’ diseases. The biggest and most exciting challenges will involve moving from “simple” problems—examining single genetic mutations in controlled systems—to more complex problems—examining multiple mutations in the context of cells’ multicellular environments. With challenges like these ahead, Theo may have found himself a career as an engineer after all. A genetic engineer, that is.
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Author’s note: The photo of Theo in the lab was taken by Noah Berger and provided to us by Theo.