The Role of CRISPR in Developing Next-Generation Antibiotics

By Dr. Luis Vaschetto, Ph.D.Reviewed by Lily Ramsey, LLM

Antibiotic resistance constitutes a major public health problem. The overuse and misuse of antibiotics have promoted the evolution of resistance in bacteria (e.g., Methicillin-resistant Staphylococcus aureus) to counteract these life-saving drugs.

This renders once-effective treatments useless, leaving us vulnerable to infections. Consequently, the need for new therapies is critical.

Fortunately, the emergence of CRISPR technology, a revolutionary gene-editing tool with immense potential, offers a new frontier in antibiotic development. This technology allows scientists to target and disable genes that confer antibiotic resistance.

Image Credit: IM Imagery/Shutterstock.com

The Antibiotic Resistance Crisis: A Looming Threat

The emergence of antibiotic resistance is driven by Darwinian selection at the microbial level. Antibiotic use selects for bacteria with mutations or acquired genes that encode resistance mechanisms, such as enzymatic degradation, enzymatic modification of targets, or efflux pumps capable of maintaining homeostasis in bacterial cells^1,2,3.

These mechanisms rapidly amplify resistant bacteria under continued antibiotic exposure, rendering traditional drugs ineffective. This creates a scenario where common infections become untreatable, increasing morbidity, mortality, and healthcare costs.

Therefore, the emergence of multidrug-resistant bacteria necessitates developing novel therapeutic strategies to combat this growing public health threat.

CRISPR: A Powerful Tool for Genetic Engineering

In 2020, the Nobel Prize in Chemistry recognized Emmanuelle Charpentier and Jennifer Doudna for their groundbreaking discovery of CRISPR/Cas, a revolutionary tool for editing genomes. This versatile and user-friendly technology has transformed research across various fields, from medicine to agriculture, making genome editing more accessible to labs worldwide⁴. This gene editing system has two primary components:

• Guide RNA: A molecule of RNA that directs CRISPR to the exact location on the DNA strand where editing needs to happen. This molecule binds to the target sequence through classical complementary base pairing rules.
• Cas Protein: An endonuclease enzyme (a protein that cuts DNA within a sequence) acts as the molecular scalpel. Guided by the RNA, the Cas protein (Cas9 is most common) cleaves the DNA at the identified location. These Cas proteins can be engineered for functions beyond cutting. For example, they can be fused with enzymes to modify the epigenome at specific locations⁵.

What is CRISPR?

Combating Resistance with CRISPR: New Strategies

There are several strategies by which the CRISPR-Cas gene editing system can be used to fight against resistant bacteria. These strategies primarily involve:

1. Bacteria mainly spread antibiotic resistance by sharing genes through horizontal gene transfer. While the CRISPR-Cas system is a natural bacterial defense immune system against foreign DNA, it can also specifically target plasmids and viruses carrying antibiotic-resistance genes. This effectively disrupts the spread of these genes via horizontal gene transfer, limiting the overall development of antibiotic resistance⁶.
2. The integrity of the bacterial envelope is crucial for both resisting antibiotics and avoiding immune defenses triggered by some antibiotics that target the bacterial membrane. Consequently, the CRISPR-Cas system may help maintain this barrier by regulating specific proteins in the membrane, ultimately allowing bacteria to better withstand damage from antibiotics⁶.

From the Lab to the Clinic: Challenges and Progress

While CRISPR holds immense potential for new antibiotic-resistance therapies, translating this promise into the clinic presents challenges^7,8. Delivering the CRISPR machinery (guide RNA and Cas9 protein) effectively into target cells -in this case, bacteria cells within the human body- remains a major hurdle.

Researchers are exploring various delivery methods, such as liposomes, nanoparticles, or viral vectors, but ensuring safe and specific delivery requires further refinement⁸. Equally important, CRISPR can cause unintended genetic sequence edits, the so-called off-target effects. Mitigating these undesired effects and ensuring the safety of CRISPR therapies in humans is crucial⁷.

Finally, bacteria can develop multiple resistance mechanisms simultaneously. Disabling just one gene might not be sufficient, necessitating strategies that target multiple resistance pathways. Developing such strategies to stay ahead of the bacterial evolutionary arms race is essential for long-term effectiveness.

Despite these challenges, the research on CRISPR-based systems is rapidly progressing. New Cas proteins with even higher target specificity are being identified and engineered^9,10.

This minimizes the risk of off-target edits and enhances the safety profile of CRISPR therapies. On the other hand, careful design of delivery systems to minimize interaction with human cells is another area of active research^11,12.

This can further reduce the potential for unintended consequences. Addressing these hurdles will be crucial in bringing this revolutionary technology to the forefront of the fight against antibiotic resistance.

The Future of Antibiotic Development: A Hopeful Outlook

With the CRISPR-Cas technology at the forefront, the recently developed gene editing tools hold immense promise for starting a new era in the fight against antibiotic resistance. While challenges in delivery, safety, and bacterial countermeasures remain, ongoing research in such technologies offers promising avenues for overcoming these issues.

As the original CRISPR gene editing system continues to evolve, with more precise tools and efficient delivery systems on the horizon, the potential for developing a new generation of highly specific and efficacious antimicrobials becomes increasingly real. The CRISPR-Cas system is a powerful tool that has the potential to create a new arsenal of effective treatments, effectively safeguarding public health for generations to come.

References    

1. Wright, G. D. (2005). Bacterial resistance to antibiotics: enzymatic degradation and modification. Advanced drug delivery reviews, 57(10), 1451-1470. https://pubmed.ncbi.nlm.nih.gov/15950313/ 
2. Schaenzer, A. J., & Wright, G. D. (2020). Antibiotic resistance by enzymatic modification of antibiotic targets. Trends in molecular medicine, 26(8), 768-782. https://pubmed.ncbi.nlm.nih.gov/32493628/
3. Huang, L., Wu, C., Gao, H., Xu, C., Dai, M., Huang, L., ... & Cheng, G. (2022). Bacterial multidrug efflux pumps at the frontline of antimicrobial resistance: An overview. Antibiotics, 11(4), 520. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032748/
4. Vaschetto LM. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases (2022). ISBN 9780367542863. Publisher: CRC Press (Taylor & Francis Group). Boca Raton, FL, USA. https://www.google.com/aclk?sa=l&ai=DChcSEwjK1J7hl9aGAxWsiVAGHTFqAIIYABASGgJkZw&ase=2&gclid=CjwKCAjwjqWzBhAqEiwAQmtgT3T4ZoLE3eiPaXZyi8QrLTVG6Fo7gecLgxCF1kffJug3ESU-IoPcnhoCMNcQAvD_BwE&sig=AOD64_1uV7JrPBbo5uPqUNkWjWKx1-bd8A&q&nis=4&adurl&ved=2ahUKEwiwwInhl9aGAxVqU0EAHa_rBXUQ0Qx6BAgGEAE  
5. Vaschetto LM (2017) Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion. Current Genetics, Springer Berlin Heidelberg. DOI: 10.1007/s00294-017-0765-9.
6. Tao, S., Chen, H., Li, N., & Liang, W. (2022). The application of the CRISPR-Cas system in antibiotic resistance. Infection and drug resistance, 4155-4168. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9356603/
7. Eduardo Rodriguez Yunta, Vaschetto LM. Political, Regulatory and Ethical Considerations of the CRISPR/Cas Genome Editing Technology. In: Vaschetto L.M (Ed.) CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases (2022). Publisher: Science Publishers CRC Press (Taylor & Francis Group). Boca Raton, FL, USA. https://www.taylorfrancis.com/chapters/edit/10.1201/9781003088516-4/political-regulatory-ethical-considerations-crispr-cas-genome-editing-technology-eduardo-rodriguez-yunta-luis-maria-vaschetto
8. Vaschetto LM. CRISPR/Cas and Gene Therapy: An Overview. In: Vaschetto L.M (Ed.) CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases (2022). Publisher: Science Publishers CRC Press (Taylor & Francis Group). Boca Raton, FL, USA. https://www.taylorfrancis.com/books/edit/10.1201/9781003088516/crispr-cas9-based-genome-editing-treating-genetic-disorders-diseases-luis-mar%C3%ADa-vaschetto
9. Chatterjee, P., Jakimo, N., Lee, J., Amrani, N., Rodríguez, T., Koseki, S. R., ... & Jacobson, J. (2020). An engineered ScCas9 with broad PAM range and high specificity and activity. Nature Biotechnology, 38(10), 1154-1158. https://pubmed.ncbi.nlm.nih.gov/32393822/
10. Kim, D. Y., Lee, J. M., Moon, S. B., Chin, H. J., Park, S., Lim, Y., ... & Kim, Y. S. (2022). Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nature biotechnology, 40(1), 94-102. https://pubmed.ncbi.nlm.nih.gov/34475560/
11. Yip, B. H. (2020). Recent advances in CRISPR/Cas9 delivery strategies. Biomolecules, 10(6), 839. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356196/
12. O’Keeffe Ahern, J., Lara-Sáez, I., Zhou, D., Murillas, R., Bonafont, J., Mencía, Á., ... & Wang, W. (2022). Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system. Gene therapy, 29(3), 157-170. https://www.nature.com/articles/s41434-021-00282-6

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