Author: Ahmed Tolba | Major: Biomedical Engineering | Semester: Fall 2024

My name is Ahmed Tolba, and I am a Biomedical Engineering student in the University of Arkansas
College of Engineering. This research was conducted under the mentorship of Dr. Chris Nelson during
the Spring 2024 and Fall 2024 semesters. I am a premed student with aspirations to combine patient care
and scientific discovery in my future career. This research focused on understanding macrophage
polarization using the CRISPR dCas9-KRAB system. We aimed to create a cell line expressing
dCas9-KRAB and clone specific gRNAs (guide RNAs) targeting the inflammatory genes IL-6 and IL1β.
Moreover, I began a new project focusing on determining the diagnostic properties of SHERLOCK on
novel viral targets in wastewater.

During my freshman year, I took an honors college gene editing forum with Dr. Chris Nelson on CRISPR
and biomedical ethics. His class made me interested in learning more about gene therapeutic technology. I
emailed Dr. Nelson, expressing my enthusiasm for his research and asking if I could join his research
group, and he kindly welcomed me into his lab. In my research, I quickly learned that macrophages, a
type of immune cell, can “polarize” based on environmental signals. Our goal was to eventually use the
dCas9-KRAB system to control this process as it pertains to inflammation. dCas9-KRAB reduces gene
activity but does not cut DNA. This allows us to study how gene expression influences cell behavior.
There are many applications of this technology in medicine. For example, in patients with diabetes,
chronic wounds are a common complication since they often fail to heal properly due to persistent
inflammation and poor macrophage function. dCas9-KRAB could in theory be used to modulate gene
expression in macrophages to reduce inflammation and promote better wound healing.

The biggest challenge to this research was likely the molecular cloning process. Cloning the specific
gRNAs, short RNA sequences that guide the system to specific locations in the genome to modify gene
expression, for IL-6 and IL1β required careful planning. There were many instances where the cloning
didn’t work, which led to delays in the project. Several other gRNAs such as select versions for TNF-α,
also failed to clone. Cloning genes into a lenti-guide backbone is a common method in genetic research,
but there are several points in the process where mistakes can happen. In genetic cloning, if there’s a
mistake in the genetic sequences or the design of our constructs, the entire process can fail, preventing
successful cloning. Another issue could be low efficiency of ligation, the process where DNA pieces are
joined together using enzymes. If the ligation conditions are suboptimal or the enzyme quality is poor, the
DNA fragments won’t stick together properly.

For Fall 2024, I started a new project focused on using SHERLOCK (Specific High-sensitivity Enzymatic
Reporter Unlocking) to develop a diagnostic method for detecting novel viral targets in wastewater
systems. SHERLOCK is a CRISPR-based diagnostic platform that uses the Cas13 enzyme to target
specific RNA sequences and produce a detectable signal, making it a highly sensitive and accurate tool
for diagnostics. This project involves a key process called recombinase polymerase amplification (RPA),
which is a method to amplify nucleic acids at a constant temperature. Unlike traditional PCR, RPA is
faster, doesn’t require expensive equipment, and can be done in simpler settings. It allows us to amplify
small amounts of viral genetic material from wastewater, which is then analyzed using SHERLOCK to
detect specific viruses. Wastewater is a valuable tool for monitoring public health because it contains
genetic material shed by infected individuals. By identifying viral sequences in wastewater, this project
could provide early detection of outbreaks, track the spread of viruses, and monitor how well public
health interventions are working. This approach is especially useful for detecting emerging viruses or
those that aren’t widely studied. Additionally, because SHERLOCK and RPA can be used in
resource-limited settings, this research could improve access to diagnostics in areas where traditional
methods are unavailable.

As a premed and engineering student, balancing classes and other commitments with research
responsibilities could be another challenge. Strong organizational planning became necessary. I sought
guidance from my PhD student mentors and Daniel Maxenburger and Dr. Allie Ivy, who helped train me
with protocols. For time management, I created a schedule and prioritized tasks to ensure I could dedicate
sufficient time to both my studies and research. Dr. Nelson’s guidance was very important in shaping the
project and helping me overcome technical challenges. I presented my research at the National
Undergraduate Research Week Symposium. Presenting allowed me to share our findings, receive
feedback, and network with other student researchers. As for what next for me–I am currently applying to
medical school and hope to integrate the research skills I have gained in the Nelson Lab in my future
career as a physician.