CRISPR, Phage Display, and Synthetic Biology: Revolutionizing the Future of Phage Therapy

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by Jorge Luis Alonso with ChatGPT-4

This article provides new insights into the rapidly developing field of phage therapy, focusing on the genetic engineering of bacteriophages (phages) to increase their precision and efficacy in treating bacterial infections. This approach is closely aligned with the principles of precision medicine, where treatments are tailored to individual patient profiles.

• Rationale. While many studies have examined the general use of phage cocktails to treat infections, fewer have examined recent advances in phage bioengineering to improve their specificity and targeted delivery. This review addresses this gap, particularly in the context of precision medicine, where treatments are becoming increasingly personalised.
• Novelty. This article reviews key innovations in phage bioengineering, such as the use of CRISPR-Cas systems, phage display technology and synthetic biology. It also highlights the potential to develop phage therapies tailored to a patient’s unique microbiome or specific infection profile. Although still an emerging field, this personalised approach holds great promise but has not yet received significant attention in the current literature.
• Relevance. With the growing focus on personalised medicine, this review highlights gaps in phage therapy, which often relies on generalised treatments. By focusing on bioengineered phages designed for targeted applications, the article contributes to the broader discussion on innovative, patient-specific therapies in line with modern healthcare trends.

Introduction: Phage therapy and the rise of personalised medicine

Antibiotic resistance is one of the most pressing public health threats of our time, driving the need for alternative treatments. In response, there has been renewed interest in phage therapy, which uses bacteriophages — viruses that specifically infect bacteria — to treat bacterial infections.

First discovered in the early 20th century, phages were initially used as a treatment before antibiotics became widely available. However, the global rise in antibiotic resistance has revived interest in phage therapy, especially as traditional antibiotics become less effective against drug-resistant bacteria.

The World Health Organization (WHO) warns that antimicrobial resistance (AMR) could lead to millions of deaths a year by 2050 if new therapies are not developed. Phages offer a potential solution due to their ability to specifically target and kill bacterial cells, as well as their ability to disrupt biofilms, which are often resistant to antibiotics.

Recent advances in genetic engineering have further expanded the potential of phage therapy. New technologies such as CRISPR-Cas systems, phage display and synthetic biology are enabling scientists to create phages with greater precision and efficacy. This fits within the framework of precision medicine, where treatments are tailored to individual patients. By customising phages to target specific bacteria within a patient’s microbiome, healthcare providers could offer more effective and personalised treatments.

In addition, phage therapy is expanding beyond the treatment of infections. Recent research suggests that phages may also have anti-inflammatory and immunomodulatory properties, which could be useful in the treatment of autoimmune diseases, allergies and complications such as graft-versus-host disease. As phage therapy continues to develop, it may play a critical role in personalised medicine approaches for a wide range of diseases.

Recent Advances in Phage Bioengineering: CRISPR, Phage Display, and Synthetic Biology

Advances in molecular biology are enabling the development of engineered bacteriophages that are more specific, adaptable and therapeutically effective. These innovations are being driven by three key technologies: CRISPR-Cas systems, phage display and synthetic biology. These approaches address the traditional challenges of phage therapy, such as limited host specificity and bacterial resistance, and position bacteriophages as a promising tool for precision medicine.

CRISPR-Cas Systems: Precision engineering for targeted therapeutics

CRISPR-Cas, originally discovered as a gene editing system in bacteria, has been repurposed to improve phage therapy. One promising application of CRISPR-Cas technology is to combat antibiotic-resistant bacteria. By delivering the CRISPR-Cas machinery via phages, scientists can disrupt genes responsible for resistance, making bacteria susceptible to existing antibiotics.

In particular, the CRISPR-Cas9 system allows phages to make precise modifications, such as inserting, deleting or altering specific genetic sequences. This technology has been used to target antibiotic-resistant bacteria, including Staphylococcus aureus and Klebsiella pneumoniae. In addition, CRISPR-Cas systems have expanded the role of phages beyond simply killing bacteria. Phages can now be engineered to carry therapeutic molecules, such as enzymes that break down biofilms, increasing their effectiveness against chronic infections.

Phage display technology: Tailoring phages for targeted delivery

Phage display technology allows scientists to modify phages to present peptides or proteins on their surface, thereby enhancing their ability to target specific bacteria. This technique is widely used in molecular biology to identify peptides or antibodies that bind with high specificity to particular targets.

In phage therapy, phage display allows the creation of phages that can recognise and bind to specific bacterial surface receptors, improving selectivity. This reduces the likelihood of off-target effects and minimises disruption to the patient’s natural microbiota. Phage display also has potential applications in vaccine development, where phages can be engineered to present antigens from harmful bacteria or viruses, thereby stimulating the immune system.

Synthetic biology: Reprogramming phages for broader application

Synthetic biology is introducing innovative methods to improve phages by extending their host range, improving their stability and enabling them to deliver therapeutic molecules. A key challenge in phage therapy is the limited host range of natural phages. However, using synthetic biology techniques such as genome editing and recombination, researchers can extend the host range of phages, making them effective against a wider range of bacterial pathogens.

One promising development is the creation of synthetic phages made from DNA fragments. These designer phages can be tailored for specific therapeutic purposes, such as delivering antimicrobial proteins or CRISPR-Cas systems to bacterial cells. This approach enables highly targeted and effective treatments, particularly against multidrug-resistant bacteria.

Precision medicine and phage therapy: Personalising treatments based on the microbiome

Microbiome profiling — the analysis of microbial communities in the human body — is revolutionising the personalisation of phage therapy. Each person’s microbiome is unique and plays a critical role in health and disease. By identifying harmful bacteria within a patient’s microbiome, clinicians can select or engineer phages that specifically target these bacteria while preserving beneficial microbes.

The role of the microbiome in health

The gut microbiome in particular influences many aspects of health, including immune function, metabolism and mental well-being. Variations in microbiome composition have been linked to conditions such as inflammatory bowel disease, autoimmune disorders and infections. Phage therapy is particularly well suited to personalised medicine because phages can be engineered to target specific bacterial strains identified through microbiome profiling, reducing the risk of collateral damage to beneficial bacteria.

Tailoring phages for precision treatment

By engineering phages to target harmful bacteria identified in a patient’s microbiome, clinicians can deliver more precise and effective treatments. This approach contrasts with antibiotics, which often kill both harmful and beneficial bacteria, disrupting the microbiome. Customised phages reduce this risk and minimise the development of bacterial resistance, making treatment both safer and more sustainable.

Challenges and future directions

Despite the potential of bioengineered phages, several challenges remain. Bacterial resistance to phages, difficulties in effective phage delivery and interactions with the immune system are key areas requiring further research and innovation. Advances in bioengineering, such as the development of immune-evading phages and the optimisation of delivery systems, will help address these challenges. In addition, regulatory hurdles need to be overcome to streamline the development and approval of phage therapies.

Conclusion

Phage therapy is being revolutionised by advances in genetic engineering and precision medicine. By tailoring phages to target specific bacterial strains, clinicians can provide more effective and personalised treatments, particularly for antibiotic-resistant infections. As bioengineering technologies advance, the integration of phage therapy into mainstream medicine is expected to increase, offering new hope for the treatment of bacterial infections and microbiome-related diseases.

References

1. Ahmed, F., Shamim, N. J., Das, A., Sharma, H. K., Grewal, A. S., & Pandita, D. (2024). Combating antimicrobial resistance: A paradigm shift from general to precision medicine. Chemical Biology Letters, 11(2), 662. https://pubs.thesciencein.org/a662/
2. Balcha, F. B., & Neja, S. A. (2023). CRISPR-Cas9 mediated phage therapy as an alternative to antibiotics. Animal Diseases, 3(4), 1–10. https://doi.org/10.1186/s44149-023-00065-z
3. Barbu, E. M., Cady, K. C., & Hubby, B. (2016). Phage therapy in the era of synthetic biology. Cold Spring Harbor Perspectives in Biology, 8(a023879). https://doi.org/10.1101/cshperspect.a023879
4. Brown, R., Lengeling, A., & Wang, B. (2017). Phage engineering: How advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophages. Quantitative Biology, 5(1), 42–54. https://doi.org/10.1007/s40484-017-0094-5
5. Caflisch, K. M., Suh, G. A., & Patel, R. (2019). Biological challenges of phage therapy and proposed solutions: A literature review. Expert Review of Anti-infective Therapy. https://doi.org/10.1080/14787210.2019.1694905
6. Chan, B. K., Abedon, S. T., & Loc-Carrillo, C. (2013). Phage cocktails and the future of phage therapy. Future Microbiology, 8(6), 769–783. https://doi.org/10.2217/fmb.13.47
7. Duong, M. M., Carmody, C. M., Ma, Q., Peters, J. E., & Nugen, S. R. (2020). Optimization of T4 phage engineering via CRISPR/Cas9. Scientific Reports, 10, 18229. https://doi.org/10.1038/s41598-020-75426-6
8. Fage, C., Lemire, N., & Moineau, S. (2021). Delivery of CRISPR-Cas systems using phage-based vectors. Current Opinion in Biotechnology, 68, 174–180. https://doi.org/10.1016/j.copbio.2020.11.012
9. Górski, A., Międzybrodzki, R., & Borysowski, J. (Eds.). (2019). Phage therapy: A practical approach. Springer Nature Switzerland AG. https://doi.org/10.1007/978-3-030-26736-0
10. Górski, A., Międzybrodzki, R., Węgrzyn, G., Jończyk‐Matysiak, E., Borysowski, J., & Weber‐Dąbrowska, B. (2019). Phage therapy: Current status and perspectives. Medicinal Research Reviews, 39(4), 849–886. https://doi.org/10.1002/med.21593
11. Gordillo Altamirano, F. L., & Barr, J. J. (2021). Phage therapy in the postantibiotic era. Clinical Microbiology Reviews, 34(4), e0002021. https://doi.org/10.1128/CMR.00020-21
12. Hess, K. L., & Jewell, C. M. (2020). Phage display as a tool for vaccine and immunotherapy development. Bioengineering & Translational Medicine, 5(1), e10142. https://doi.org/10.1002/btm2.10142
13. Housby, J. N., & Mann, N. H. (2009). Phage therapy. Drug Discovery Today, 14(11–12), 536–540. https://doi.org/10.1016/j.drudis.2009.03.006
14. Jaroszewicz, W., Morcinek-Orłowska, J., Pierzynowska, K., Gaffke, L., & Węgrzyn, G. (2022). Phage display and other peptide display technologies. FEMS Microbiology Reviews, 46(2), 1–25. https://doi.org/10.1093/femsre/fuab052
15. Lenneman, B. R., Fernbach, J., Loessner, M. J., Lu, T. K., & Kilcher, S. (2021). Enhancing phage therapy through synthetic biology and genome engineering. Current Opinion in Biotechnology, 68, 151–159. https://doi.org/10.1016/j.copbio.2020.11.003
16. Liu, D., Van Belleghem, J. D., de Vries, C. R., Burgener, E., Chen, Q., Manasherob, R., Aronson, J. R., Amanatullah, D. F., Tamma, P. D., & Suh, G. A. (2021). The safety and toxicity of phage therapy: A review of animal and clinical studies. Viruses, 13(7), 1268. https://doi.org/10.3390/v13071268
17. Melo, L. D. R., Oliveira, H., Pires, D. P., Dabrowska, K., & Azeredo, J. (2020). Phage therapy efficacy: A review of the last 10 years of preclinical studies. Critical Reviews in Microbiology, 46(2), 114–133. https://doi.org/10.1080/1040841X.2020.1729695
18. Międzybrodzki, R., Borysowski, J., Weber-Dąbrowska, B., & Górski, A. (2012). Clinical aspects of phage therapy. Advances in Virus Research, 83, 73–118. https://doi.org/10.1016/B978-0-12-394438-2.00003-7
19. Patey, O., McCallin, S., Mazure, H., Liddle, M., Smithyman, A., & Dublanchet, A. (2019). Clinical indications and compassionate use of phage therapy: Personal experience and literature review with a focus on osteoarticular infections. Viruses, 11(1), 18. https://doi.org/10.3390/v11010018
20. Pierzynowska, K., Morcinek-Orłowska, J., Gaffke, L., Jaroszewicz, W., Skowron, P. M., & Węgrzyn, G. (2024). Applications of the phage display technology in molecular biology, biotechnology and medicine. Critical Reviews in Microbiology, 50(4), 450–490. https://doi.org/10.1080/1040841X.2023.2219741
21. Sunderland, K. S., Yang, M., & Mao, C. B. (2017). Phage-enabled nanomedicine: From probes to therapeutics in precision medicine. Angewandte Chemie International Edition, 56(8), 1964–1992. https://doi.org/10.1002/anie.201606181
22. Wu, V. C. H. (Ed.). (2023). The gut microbiome: Bench to table. CRC Press. https://doi.org/10.1201/b22970