Content Number: 27
Author Name: Rimsha Arif
Author I’d: SBPWNC – A27
Educational Institution: Women university Multan, Pakistan
Content Title: Phage Therapy Meets CRISPR: A New Frontier in Infectious Disease Treatment
The number of the accessible therapeutics for bacterial infections has decreased due to the rapid global spread of antimicrobial resistance. Many prokaryotes have an adaptive immune system termed CRISPR-Cas, which may be engineered to target bacterial genomes and induce cell death. An promising strategy for tackling antibiotic resistance is the therapeutic repurposing of the CRISPR-Cas system. However, in order for the CRISPR-Cas system to reach the bacterial genomes, this technique needs appropriate vectors. A promising replacement for cargo delivery vectors has been offered by engineered phages.
Phage therapy: A Natural Weapon Against Bacteria
With an estimated 10:31 phages worldwide, phages are the most widespread and abundant organisms on the planet. Phages are viruses that infect bacteria. From the initial investigations demonstrating that DNA is the genetic material of living cells using phages as a model system to the discovery and implementation of temperate bacteriophage-based genetic integration tools such as lambda red recombinases and the generation of the gene-editing system RM/CRISPR, the study of phages and their interactions with their bacterial hosts has been an important aspect in the development of molecular biology.
Mechanism of Action
With the aim to reproduce vertically from mother to daughter cell, phages generally bind to specific receptors on the bacterial cell surface, inject their genetic material into the host cell, and either integrate this material into the bacterial genome (in the instance of “temperate” phages) or employ the bacterial replication machinery to produce the next generation of phage progeny and lyse the cell (in the the event of “lytic” phages). Lysogenic phages, like in contrast to lytic phages, incorporate their genetic material into the bacterial chromosome as an endogenous prophage (phage DNA can be scattered throughout bacterial generations as a plasmid, nevertheless this is less common). The prophage is then dispersed by the bacterial lysogen with each cell cycle.A transition to the lytic cycle and the release of phage progeny into the environment can be attributed to environmental stressors on the bacterial host, that may activate the lysogenic phage from the latent prophage form.
Limitations of Phage Therapy
Multidrug-resistant (MDR) strains of many hazardous bacteria have developed as an outcome of the widespread administration of antibiotics globally.A promising strategy against MDR is phage therapy, which employs phages that specifically infect and kill the host bacteria without affecting other bacteria (Rodriguez-Gonzalez et al., 2020; Kim et al., 2021; Salazar et al., 2021; Jia et al., 2023). However, most phages’ restricted host range and potent host specificity make them inappropriate for use in phage therapy.
There are two primary reasons for the emergence of these limitations. First, host bacteria can use protections like immunization or evolution to prevent infection by phages with a narrow host range. The target pathogenic bacteria acquires phage resistance as a result of this evasive behavior. Second, even within the same species, there are significant variations in clinical isolates of bacterial pathogens. Due to this, a phage that’s effective for one patient might not work for another if it’s unable to infect and eliminate both bacterial strains.
Applications of Phage Therapy
Phage therapy presents the potential to treat persistent medical conditions where bacteria perform an integral part in pathogenesis, besides to the therapeutic use of phages to treat bacterial infections. By activating prophages with medicines or nutritional products like Stevia rebaudiana and bee propolis extracts, phages can also be employed to groom the gut flora.This method of modifying the composition or function of bacteria has been separate from phage therapy and was lately called “phage rehabilitation.”It was found that 118 phage considerably suppressed the bio-burden of several Salmonella enterica serotypes whenever it was administered orally to broiler chickens with antacid protection at the beginning of the 21st century. Preparations of the phages could be used instead of or in addition to antibiotics before slaughter in pigs, cows, and poultry to stop an array of food-borne bacterial infections from spreading into the food chain. Additionally to their uses in food safety, phages may be utilized in the built environment, such as in hospitals, to detect bacteria that are resistant to various drugs and to disinfect surfaces.
CRISPR Technology: Precision Gene Editing
Clustered regularly interspaced short palindromic repeats, or CRISPR (/ˈkrɪspər/), are a family of DNA sequences that are found in the genomes of prokaryotic organisms including bacteria and archaea. A DNA fragment from a bacteriophage that previously infected the prokaryote or one of its predecessors is the origin of each sequence found in an individual prokaryotic cell. During subsequent infections, these sequences are used to recognize and eradicate DNA from similar bacteriophages. As an outcome, these sequences are crucial for prokaryotes’ antiviral (or anti-phage) defensive mechanism and provide a kind of acquired, heritable immunity. Approximately 90 percent of sequenced archaea and 50% of sequenced bacterial genomes possess CRISPR.
CRISPR Lexicon
Guide RNA (gRNA) is a type of ribonucleic acid (RNA) molecule that binds to Cas9 and shows, based on the sequence of the gRNA, the location at which Cas9 will cut DNA. Cas9 is an enzyme referred to as a CRISPR-associated (Cas) endonuclease, or enzyme, that works as “molecular scissors” to cut DNA at a particular position.
How does it work?
It is similar to the immune system of humans. We develop an excessive amount of antibodies for an immune memory when we get infected by a virus. These antibodies then quickly recognize and destroy invaders when the same virus infects us once again. CRISPR assists in the development of a genetic memory when a virus infects a bacterial cell. A small portion of the virus’s genome is captured by the bacteria, which then inserts the DNA into its own. CRISPR creates a new “guide RNA” from that newly gained DNA sequence, which helps CRISPR in detecting the invader with sequence complementarity (A binds to T and C binds to G).Hence, the guide RNA rapidly recognizes the virus the next time it infects that bacterial cell.
Limitations
Though CRISPR/Cas is a highly efficient technique, it has major drawbacks. Large-scale delivery of the CRISPR/Cas material to mature cells is tricky, which appears to be an issue for many clinical applications. The most common approach of transportation is through viral vectors. It is not completely productive, hence even cells that ingest CRISPR/Cas might not be able to change their genomes. It is not completely accurate, and although “off-target” modifications are uncommon, they can have serious repercussions, especially in therapeutic settings.
Application’s
CRISPR-Enhanced Phage Treatment
Researchers are now collaborating to create CRISPR-modified phages to improve their effectiveness as antibacterial drugs. To allow the Cas enzyme to target a specific bacterial (as compared to viral) DNA sequence, scientists can create distinctive guide RNA sequences in the lab. The CRISPR-Cas system can be transcribed by incorporating it into the phage’s genome after it has been developed. Each phage will have the CRISPR-Cas system sequence besides to its normal genetic material, that is injected into the targeted bacteria causing it to break down and disperse the phage further.
Two different enzyme classes that are a component of the CRISPR system, Cas9 and Cas3, are being studied for their ability to develop antibacterial characteristics. Johnson & Johnson.35 subsidiary Janssen Pharmaceuticals is working on one such CRISPR-phage antibacterial.The pharmaceuticals, known to be the most developed in development for a drug of its kind, entered Phase 1 clinical trials in 2020 for use against Escherichia coli. Phage genetically engineered to incorporate CRISPR-Cas has been demonstrated in clinical trials to be more effective than naturally occurring (or “wild-type”) phage in eliminating Clostridium difficile, a bacterium species which is vulnerable to antibiotic resistance.By increasing eradication by several orders of magnitude and minimizing the likelihood of resistance, CRISPR-Cas3 improves the bactericidal effect of phage. Because the CRISPR-Cas system is ruining the bacterial cell’s essential genes as the phage multiplies within the cell, CRISPR-phage kill host bacteria more quickly. This might result in cell death before phage-mediated biochemical processes cause cell lysis.
Gross structural defects of the host nucleus, such as the formation of micronuclei (damaged chromosome fragments or whole chromosomes erroneously left outside the nucleus during cell division), may be a consequence of CRISPR-Cas editing. These flaws initiate a mutational procedure referred to as chromothripsis, which can result in cancer.
References
1) Pacia, D. M., Brown, B. L., Minssen, T., & Darrow, J. J. (2024). CRISPR-phage antibacterials to address the antibiotic resistance crisis: scientific, economic, and regulatory considerations. Journal of Law and the Biosciences, 11(1), lsad030.
2) Jia, H. J., Jia, P. P., Yin, S., Bu, L. K., Yang, G., & Pei, D. S. (2023). Engineering bacteriophages for enhanced host range and efficacy: Insights from bacteriophage-bacteria interactions. Frontiers in Microbiology, 14, 1172635.
3) Lin, D. M., Koskella, B., & Lin, H. C. (2017). Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World journal of gastrointestinal pharmacology and therapeutics, 8(3), 162.4
4) Strathdee, S. A., Hatfull, G. F., Mutalik, V. K., & Schooley, R. T. (2023). Phage therapy: From biological mechanisms to future directions. Cell, 186(1), 17-31.
6) Fage, C., Lemire, N., & Moineau, S. (2021). Delivery of CRISPR-Cas systems using phage-based vectors. Current opinion in biotechnology, 68, 174-180.
7)https://news.stanford.edu/stories/2024/06/stanford-explainer-crispr-gene-editing-and-beyond
8) https://en.m.wikipedia.org/wiki/CRISPR
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