The Future of Lyme Disease and Borrelia burgdorferi Research is Looking Clearer and CRISPR Than Ever Before

By: Skyler Odin '21

The development of a new clustered regularly interspaced palindromic repeats interference (CRISPRi) platform has just sped up and revolutionized the study of Lyme Disease and its causative bacteria Borrelia burgdorferi forevermore.

According to the Centers for Disease Control and Prevention (CDC), Lyme Disease, the most common vector-borne disease in the United States, is becoming even more widespread and common. Vector-borne diseases are illnesses caused by pathogens and parasites that are transferred to humans by vectors (blood-feeding arthropods like ticks and mosquitos). Lyme disease is caused by the infection of a bacteria called Borrelia burgdorferi and is transmitted via the bite of an infected blacklegged tick.

CDC: Comparison of reported cases of Lyme Disease from 2001 to 2018. Image source

Blacklegged ticks (or deer ticks, Ixodes scapularis) are found in the northeastern, mid-Atlantic, and north-central United States. Western blacklegged ticks (Ixodes pacificus) are located on the Pacific Coast. Ticks can attach to any part of your body but are often found in hard-to-see areas like the groin, armpits, and scalp. In most cases, the tick must be attached for at least 36 to 48 hours before the Lyme disease bacterium, B. burgdorferi, can be transmitted. Typical symptoms of Lyme Disease include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. Symptoms can worsen when left untreated, leading to joint pain, heart problems, and some other neurological problems.

Blacklegged (Deer) Ticks. Image Source

B. burgdorferi is a long and cylindrical gram-negative helically-shaped spirochete bacterium. A single cell usually measures 1 μm wide, but they can be up to 10-25 μm long. Spirochete bacteria have a unique structural characteristic where the bacteria's flagella are located inside the periplasm, the space between the bacteria's inner and outer cell membranes. The interactions between the flagella and cell cylinder allow the cell to travel through highly viscous fluids and materials (like human tissue) causing it to be highly invasive.

Electron Micrograph of Borrelia burgdorferi Image Source

Research surrounding B. burgdorferi has been gaining traction and importance with the rise in cases in addition to the fact that the bacterium’s mechanisms and pathological pathways are still a relative mystery. The problem is results are so slow-moving due to the importance of genetic tractability in studying the biology of a pathogen. Genetic tractability refers to how amenable an organism is to genetic manipulation. Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders microbiology research and development beyond a few model organisms. B. burgdorferi has an extraordinarily unusual genome that is highly segmented and predominantly linear which renders it fairly genetically intractable. Creating unique mutants for each specific experiment is incredibly tedious and time-consuming, but thanks to a talented group of researchers, there might be some relief and new data not so far in the future.

A paper by Takacs et. al. was recently published this year describing a new “CRISPR Interference Platform for Selective Downregulation of Gene Expression in Borrelia burgdorferi.” The paper outlines the development of a clustered regularly interspaced palindromic repeats interference (CRISPRi) platform created to speed up B. burgdorferi research by providing a quick and effective way to genetically edit and manipulate B. burgdorferi. CRISPR is a revolutionary gene-editing tool developed by Jennifer Doudna and Emmanuelle Charpentier in 1987. The paper employs a CRISPR interference platform that uses a dCas9 protein (Cas-9 proteins are used by CRISPR and many other gene-editing platforms) and a customizable single guide RNA (sgRNA). The dCas9-sgRNA complex binds to DNA elements complementary to the specific sgRNA chosen and causes a steric block that interferes with transcription by RNA polymerase and results in the repression (turns off) of the target gene. The dCas9-sgRNA complex renders the CRISPRi highly specific and, to sweeten the deal, the researchers designed over 30 CRISPRi plasmids (genetic structures in a cell that can replicate independently of the chromosomes, typically used in the laboratory manipulation of genes) to facilitate studies of various genes in wild-type and genetically modified strains of B. burgdorferi.

CRISPR Interference: The dCas9-sgRNA complex effect depends on the addition of IPTG (effector domain) because IPTG controls the expression of the dCas9 protein. The addition of IPTG induces the expression of dCas9, creating the dCas9-sgRNA complex, resulting in the repression of the target gene. Image Source

The team tested the functionality of their platforms by targeting genes connected to the bacteria’s motility (movement) and cell morphogenesis (cell size and shape) flaB, mreB, rodA, and ftsI. By specifically selecting these genes, the researchers clearly saw observable changes in the bacterial cells and could easily prove the mutants created using the CRISPRi platform were successful. These tests produce promising results (efficiencies of at least 95%) and incredibly captivating microscopy images. During testing, the researchers controlled the expression of dCas9 through the omission or addition of isopropyl-b-D-thiogalactopyranoside (IPTG). In the absence of IPTG (uninduced condition), cells of B. burgdorferi CRISPRi strains had their dCas9 expression repressed (this would result in no dCas9-sgRNA complex present in the cells). When IPTG was added (induce condition), the bacteria expressed dCas9, which resulted in down-regulation of gene expression (repression of the target gene).

One of the most impactful examples of microscopy in the Takacs et. al. article is Figure 4. It dives into the idea of using the CRISPRi platform to create a mutant of B. burgdorferi repressing the FlaB gene responsible for directing flagellin growth. Flagella are the core of B. burgdorferi’s motility and flat wave morphology. Figure 4A’s inverted dark-field images show the control strain expressing the FlaB regardless of the addition of IPTG. You can see that the bacterial cells have retained their helical shape and showed no difference in motility. Figure 4B’s inverted dark-field images compare the difference between cells expressing FlaB (no IPTG) and cells lacking FlaB (IPTG, outlined in red). The cells have straightened out and shown a partial loss of motility during observation. Figure 4C and Figure 4D are Cryo-electron tomography images depicting the reason why FlaB depletion resulted in cell straightening and partial motility loss: In the 3D model shown in Figure 4C, you can see how the filaments (flagella) are wrapped around the cell body depicted in green, while they are missing in Figure 4D’s 3D model. The unique way the flagella are wrapped around the cell body gives rise to B. burgdorferi’s helical shape and especially effective (and invasive) manner of movement.

Figure 4: Phenotypic characterization of flagellin depletion. Loss of flagella resulted in partial loss of motility and cell straightening. Image Source

The results are shining examples of the usefulness of the presented CRISPRi platform approach. It produced efficient gene product down-regulation, was user friendly, and had relatively fast clone generation. In other words, the platform was highly successful in providing a quick and effective way to genetically edit and manipulate B. burgdorferi, removing the barrier of genetic intractability and significantly speeding up research surrounding B. burgdorferi and Lyme Disease. It is important to keep in mind that this platform is not a replacement for traditional homologous recombination-based genetic research methods, but it does provide efficient advantages when used in tandem with traditional methods due to its procedural ease, speed, efficiency, and scalability. I, for one, am excited to see the future research and data surrounding B. burgdorferi and Lyme Disease, created through the marriage of the two genetic research approaches!