Alternative Selectable Markers
Our initial idea was to have our alternative selectable markers utilized inside and outside scientific labs to provide an antibiotic resistance-free way to check for successful transformants. Because introducing antibiotic resistance genes into bacteria can further contribute to the growing healthcare crisis that is antimicrobial resistance, it is against the law to use these constructs outside of the lab. Our selectable markers that rely on metabolic genes can be more widely used in the scientific community as they would be permitted to leave the lab without the worry of spreading antibiotic resistance. This would give scientists an opportunity to test their constructs in more of a real world setting, as opposed to just a lab. Through this portion of the project, we hoped to contribute new and safe tools to the scientific community.
Team UFlorida is excited about the utilization of CRISPR in mitigating antibiotic resistance, as it has potential to target a wide variety of pathogens. The CRISPR target can be easily modified by exchanging the guide RNA, giving this system the ability to target a plethora of diseases. Team UFlorida focused on diseases of the gut microbiome and the genital tract. Physicians are reliant on antibiotics, such as tetracyclines and ceftriaxone, to target pathogens of the gut microbiome and genital tract respectively. Due to the yearly 10 million deaths that antibiotic resistant infections are projected to cause by 2050, it is imperative to have therapies in place when antibiotics are no longer effective, and that can reduce this risk without having to give up currently implemented antibiotics.
CRISPR - Conjugation System in the Gut
Being highly specific to their targets, bacteriophages offer great potential to deliver CRISPR to a target pathogen. Since our lab does not have the means to work with viruses, we turned to a system that could deliver CRISPR using plasmids. Our main concern brought up by our PI was the absence of an evolutionary benefit. If these plasmids do not offer bacteria an evolutionary benefit, we cannot be sure the target bacteria will take up the CRISPR plasmid. We then turned to the 2019 SDU Denmark team, as they figured out a way to utilize a two plasmid system to deliver CRISPR to their targets, without the concern of bacteria not taking up the plasmids. Our wet lab team met with the two team captains, Stine and Annemette, from the 2019 SDU Denmark team to further discuss the details of their project. They first told us to try to integrate CRISPR components on a conjugative plasmid, such as the R or F plasmid. This way, transformation could be more efficient, as all of the necessary genes are on one plasmid, as opposed to two. Our PI told us that conjugative plasmids are already very big, so adding CRISPR components would make the assembly of this plasmid very difficult. So, we turned to a two plasmid system - a CRISPR plasmid and a conjugative plasmid. Instead of targeting GFP like team SDU Denmark, we would target a bacterial chromosome when testing our system, and when implementing this in a clinical setting. Also while testing this system in the lab, we would use our alternative selectable markers, as opposed to antibiotic resistance markers, to see if the target bacteria took up our plasmids.
SDU Denmark 2019 team captains explaining their project in more depth during the zoom meeting
They also shared information that helped with the end goal of the project. They told us that a lot of gut bacteria thrive on sharing genes, so our CRISPR plasmid would likely be taken up by bacteria in that environment. We shared with them our concern about the absence of an evolutionary benefit, and they did not see that as an issue in their lab work. Therefore, we see our system being utilized in the gut microbiome to cut pathogenic bacteria that are resistant to antibiotics. We envision this therapy being orally administered in a pill that can bypass the acidity of the stomach to reach the gut. After the therapy is administered and the CRISPR is delivered, antibiotics could now be used as bacteria would be resensitized. Using this therapy would make resistant bacteria more susceptible to antibiotics that are already on the market, reducing the need for new pharmaceuticals.
Dr. Maurelli from the Emerging Pathogen Institute at UF suggested we look at ultimately targeting a gut pathogen that does not invade cells in the gut so that our donor bacteria can come into contact with the pathogen. He suggested that this therapy could be used to target Vibrio cholerae, a bacteria that is non-invasive to gut epithelium. We envision this therapy to be implemented if a patient does not respond well to antibiotics. Dr. Maurelli enforced this by noting that doctors do not have time to test for antibiotic resistance - they only treat as drugs fail.
CRISPR - Conjugation System in the Reproductive Tract
Dr. Maurelli also suggested that this therapy be used to target pathogens in the reproductive tract. He suggested targeting Gonorrhea due to its specific markers and its natural ability to exchange genetic material through other Neisseria. These transformative events are triggered by specific sequences on the plasmid, allowing for increased specificity. The fact that neisseria are naturally transformable makes them even more susceptible to antibiotic resistance, as they can easily exchange genes. Due to the specificity of Neisseria and the transformative environment of the genital tract, this therapy would also eliminate the need for a conjugative plasmid - the CRISPR plasmid could be delivered straight to the genital tract through a cream. The bacteria will then undergo transformation with other bacteria in the genital tract to deliver CRISPR to our target.