Abstract
89,000 cases of Lyme disease were reported to the CDC in 2023, yet experts estimate that nearly half-a-million Americans are infected with Borrelia burgdorferi annually. This significant underdiagnosis is due to the rapid dissemination of the bacteria into tissue, causing current tests to rely on antibodies produced weeks post-infection. However, the outer surface protein, CspZ, persists in the bloodstream, allowing for early detection through LANCET.
Our diagnostic workflow begins with proximity-dependent ligation to detect CspZ and produce DNA, which is then amplified by recombinase polymerase amplification. Cas12a targets this DNA, providing a user-friendly output through a lateral flow assay.
Even with improved diagnosis, treatment continues to rely on antibiotics, contributing to antimicrobial resistance and failing to address post-treatment Lyme disease. LANCET offers a novel approach through a multiplexed CRISPR-interference system to silence critical genes, effectively killing the bacteria. Our initiative also includes community education, fostering proactive behaviors against Lyme.
Defining the Problem
Lyme disease is the most common vector-borne disease in the United States of America with an estimated 476,000 cases annually (Mead, 2022). Despite its prevalence, Lyme remains severely underdiagnosed with studies showing that 23% of distinct bullseye rashes are missed by healthcare providers, and 54% of patients without the rash receive an incorrect diagnosis (Aucott et al., 2009). Currently, there is no early diagnostic for Lyme disease, only an antibody based two-tiered serologic test that can not be used until at least two weeks post infection. Delayed or missed diagnosis, can have a significant impact on the quality of life for patients.
These widespread misdiagnoses can leave patients with years of unexplained symptoms,often furthering the prevalence of Chronic Lyme Disease or post-treatment Lyme Disease (PTLD). An estimated 15% of patients are thought to face this debilitating condition involving persistent fatigue, cognitive impairments, and chronic musculoskeletal pain, severely impacting quality of life. Due to the lack of universal diagnostic criteria for PTLD, this percentage is extremely difficult to estimate leaving it highly uncertain (Maksimyan, 2021).
Reported in all 50 states and over 65 countries worldwide, Lyme disease is quickly becoming a pressing global health issue. Due to the changing climate, average temperatures and humidity are on the rise and the scope of lyme-vulnerable populations are increasing (Bay Area Lyme Foundation, 2014). This expansion is driven by warmer temperatures that allow ticks to survive in previously unsuitable regions while expanding their active season (Couper et al., 2022). In these newly endemic regions, clinicians’ lack of experience with Lyme exacerbates both the risk of misdiagnosis and inadequate preventative measures, widening an already critical public health gap (Johns Hopkins Lyme Disease Research Center, 2019).
Pathophysiology
| Category | Details | |
|---|---|---|
| Pathogen Type | Spirochete bacterium (Borrelia burgdorferi) | |
| Location & Prevalence | Northeastern & Midwestern US | |
| Primary Hosts | Rodents and deer | |
| Tick Vectors | Black-legged deer tick (Ixodes scapularis) – NE, Mid-Atlantic, North-Central US Western black-legged tick (Ixodes pacificus) – Pacific Coast US | |
| Transmission | Spread via saliva of infected ticks during feeding | |
| Stage of Concern | Nymph stage ticks cause most human infections | |
| Feeding Requirement | Requires 36–48 hours of tick attachment for transmission | |
| Peak Season | Late spring → early fall | |
| Typical Habitat | Forests, shrublands, tall grasses |
Symptoms
Lyme disease symptoms can manifest from as early as three days or as late as a year after infection. Because they are highly variable, these symptoms are often disregarded or misdiagnosed as other illnesses (Penn Medicine, 2025). While symptoms are typically divided by stages, patients experience a variety of signs with many never experiencing the early-stage (see Fig. 1).
Current Solutions
Early and accurate diagnosis is critical for the effective treatment of Lyme disease.
Clinical diagnosis relies on the presence of the erythema migrans (EM) rash, however, it is only present in 70-80% of those infected and varies significantly in size and shape, making identification challenging (Aucott et al., 2012). Physicians also consider a patient’s exposure history to tick bites and high-prevalence areas to aid in diagnosis, though this approach lacks specificity (CDC, 2024).
Laboratory diagnostics use a two-tiered serological testing approach, beginning with an immunoassay followed by a Western blot, to detect antibodies produced by the immune system against B. burgdorferi (Johns Hopkins, 2023). These antibodies take several weeks to develop, and as a result, tests in early stages frequently lead to false negative results. Tests are often then repeated three to four weeks later, further delaying treatment.
As infection continues, the severity of the disease increases highlighting the need for a reliable and rapid detection method.
Our Approach
Lambert iGEM designed a multifaceted solution to combat Lyme disease focusing on three approaches: promoting proactive measures through education, accelerating diagnosis times, and improving treatment outcomes.
Education
During our conversations with Dr. Nicole Baumgarth, she emphasized the critical lack of awareness surrounding Lyme disease prevention and recognition. This insight shaped our Human Practices initiatives throughout the year, focusing our outreach on educating students and the broader community about Lyme disease. Targeting a diverse range of populations, we aimed to empower individuals to take proactive steps in preventing tick bites, identifying early symptoms, and seeking timely medical attention (see Education).
Diagnostic
Our diagnostic system consists of four primary systems. First, proximity-dependent ligation, detects the CspZ outer surface protein by binding aptamers and generating a DNA product. This product is then amplified by Recombinase Polymerase Amplification (RPA), a highly sensitive isothermal DNA amplification method enabling rapid, accurate amplification. The amplified DNA is detected by the Cas12a system causing the collateral cleavage of a reporter mechanism. The output is a lateral flow assay providing a user-friendly visual readout. As there is a lack of integrated RPA primer and Cas12a crRNA combined generation software, we created CASPER, an open-source tool for other researchers and iGEM teams (see Diagnostics Overview).
Therapeutic
Building upon our 2024 project SHIELD, we continued to utilize a CRISPR-interference (CRISPRi) system, consisting of a deactivated Cas9 protein and sgRNA, to downregulate critical genes in resistant bacteria. As a part of LANCET, the system was redesigned to incorporate multiplexing, targeting the critical genes Bb0250 and Bb0841 in the Borrelia burgdorferi bacteria (see Therapeutics Overview)
