Combatting Antibiotic Resistance: Gene Editing and Phages in the Post-Antibiotic Era
Picture a world where antibiotics have lost all effectiveness against bacterial infections. These antimicrobials save an estimated 200,000 American lives each year — but it is estimated that instead, in 2050, multi-drug resistant bacteria could be responsible for the death of 10 million people.
This is antibiotic resistance. And it is a true global crisis.
Now, what if I told you that gene editing, with the help of a virus named bacteriophage, can be the solution to conquering antibiotic resistance?
In this article, as a follow-up to my previous article, Phage Therapy: The Solution to Antibiotic Resistance, I will investigate the use of phage-mediated gene editing in antibiotic-resistant bacterial strains.
Antibiotics
Antibiotics stand as one of humanity’s most remarkable breakthroughs. With over 100 types, all deployed to fight a wide spectrum of bacterial infections, these curative agents have saved many lives.
The primary mode of action for antibiotics is their ability to bring about the death of bacterial cells, a process known as bactericidal action. This is achieved by obstructing the cell’s growth and replication through the inhibition of key processes such as:
- DNA synthesis: ex. Quinolone antibiotics cause DNA breaks that prevent it from replicating properly.
- Cell wall synthesis: ex. Beta-lactam antibiotics weaken the cell walls.
- Protein synthesis: ex. Tetracycline and aminoglycoside antibiotics disrupt protein production, crippling the bacterium’s growth and reproduction.
By disrupting these processes, the antibiotics can dismantle the infecting bacteria. Most of the time…
In Our Post-Antibiotic Era?
Overuse of antibiotics in the healthcare world and agriculture has led to the prosperity of certain bacterial strains. These bacteria thrive due to their acquisition of a beneficial mutation: antimicrobial resistance gene(s). This creates superbugs.
To the same degree that antibiotics have saved humans time and time again, a complete takeover of superbugs will result in a staggering number of deaths due to MDR bacterial infections.
For more information, take a look at my previous article.
What is CRISPR-Cas9?
In the history of scientific discovery, few breakthroughs have had as profound an impact on genetics and biotechnology as CRISPR-Cas9. This revolutionary technology, often called “genetic scissors,” allows scientists to edit DNA precisely, opening up unprecedented opportunities to manipulate the genetic code of living organisms.
CRISPR, short for “Clustered Regularly Interspaced Short Palindromic Repeats,” is a sequence in the bacterial genome, originally discovered by Japenese scientist Yoshizumi Ishino and colleagues while studying the bacterium Escherichia coli (E. Coli). In 1995, Francisco Mojica and other laboratories concluded that the CRISPR system, which they were studying in prokaryotes (bacteria and archaea), was a part of the prokaryotic immune system. They observed that the CRISPR loci in these organisms contained unique sequences known as “spacers” that were separated by short palindromic repeats. The spacers were derived from past encounters with foreign genetic material, such as viruses, and effectively served as a genetic memory of past viral invasions.
The next time that virus or a related virus tries to infect, the bacteria produces RNA segments from the CRISPR arrays that tether to specific regions on the virus’ DNA. An enzyme or protein such as Cas9 then cuts the viral DNA, disabling the virus.
How does this apply to us?
Using this knowledge of the naturally occurring process in which bacteria edit their genome to defend against viral infections, scientists have adapted this system to be used in the editing of DNA.
When CRISPR is inserted into a cell, it uses a guide RNA to recognize the target gene. This segment binds itself to the sequence of genes. This process imitates the RNA produced by bacteria. Similarly, along with the gRNA, there is a protein or enzyme attachment. There are a few different types of enzymes that can be used, but among the more common is Cas9. The Cas9 protein is an endonuclease that creates a double-stranded break of the DNA. Then, using the cell’s repair machinery, the DNA sequence is customized by adding, removing, or replacing a segment in the genetic information.
The protospacer adjacent motif (PAM) sequence is a short sequence (2–6 nucleotides) that follows the target DNA sequence. It is necessary for the precise cleavage by a Cas nuclease. The PAM (in addition to the gRNA) is thus needed to ensure that the Cas does not edit the wrong nucleotides.
The Role of CRISPR- Cas9 in Fighting Antibiotic Resistance
While other solutions such as alternatives like phage therapy attempt to imitate the bactericidal ability of antibiotics, a more precise and innovative approach is emerging through the use of CRISPR-Cas9 technology. Instead of replacing the use of antibiotics entirely, this method aims to directly target the root cause of antibiotic resistance, offering a promising solution to one of the most pressing challenges in modern medicine.
As mentioned earlier, antibiotic resistance arises from genetic mutations within bacteria. CRISPR-Cas9, along with its guide RNA (gRNA), is employed to identify and target antibiotic-resistance genes or sequences within bacterial strains.
Let’s break it down:
- Scientists identify the sequence of DNA that is to be changed (in the case of fighting antibiotic resistance: scientists would identify the sequence of DNA responsible for their resistance against antimicrobials).
- They create a strand of gRNA that corresponds to the nitrogenous bases of the DNA strand.
- The gRNA is attached to the Cas9 protein and this complex is introduced to the target cells (MDR bacteria).
- The gRNA locates its target and cuts the DNA strand.
- Scientists can now modify genetic information by inserting or deleting certain parts.
Numerous genes are responsible for antibiotic resistance in bacteria. Using this method, we can precisely target a couple of these genes to resensitize the bacteria to certain groups of antibiotics.
Introducing: Phage-Mediated Gene Editing
To effectively deliver the CRISPR-Cas9 system to the bacteria to do its thing, we need a vector. A vector is a vehicle used to carry a particular DNA segment (or CRISPR in this case) into a host cell.
Bacteria Eater
While several different types of vectors exist to do this, a groundbreaking approach to delivering the package arrives in the shape of bacteriophages. Bacteriophages (quite literally meaning “bacteria eater”) are viruses that only infect bacterial cells, not human cells. They are the most abundant biological agent on earth and exist in many forms: icosahedral phage (corticovirus), head-tail phage (T7), or filamentous phage (Inovirus). Phages are almost everywhere, the soil, the oceans, the sewage, etc.
How does it work?
In the context of our gene editing endeavour, engineered bacteriophages are designed to carry the Cas9 protein and the gRNA directly to the antibiotic-resistance genes of the target bacteria upon infection. These modified phages selectively infect the target bacteria, and the Cas9 protein cuts the antibiotic resistance genes or other critical sequences within the bacterial genome. This genetic sabotage incapacitates the bacteria’s ability to survive and reproduce, effectively eliminating antibiotic-resistant strains. The immune system or additional phages can then clear the remnants.
Let’s imagine this process like a heist. The vault (bacteria) is selected and the appropriate tool(s) to access the vault (bacteriophage) is/are chosen. Think of it like a screwdriver with a hex tip trying to access a panel that was screwed in with a flat screwdriver tip. In this analogy, the person harnessed to the string and slowly lowered into the vault represents the CRISPR-Cas9 protein and plucks the treasure or money (target gene: antibiotic resistance gene) from the vault. Leaving behind everything else in the vault untouched and unaffected by the heist.
By specifically targeting antibiotic resistance genes within bacterial strains, this approach can significantly reduce the selective advantage of resistant bacteria, slowing down the natural selection process. Moreover, it is highly adaptable, ensuring that we can stay one step ahead of antibiotic resistance, leaving no room for its resurgence.
Why phages?
Well, that’s a good question. There are quite a few other methods/vectors that could be selected to accomplish this job.
Below is a comparison table between the utilization of bacteriophages and three other delivery methods being explored for the delivery of CRISPR.
As you may notice, each method has its advantages and disadvantages. However, at the moment, due to their high specificity (tropism towards bacteria), cargo capacity, and safety, bacteriophages are a good place to start in the development of this method.
Considerations:
While this is an exciting topic and seems relatively simple in theory, there are still many flaws to be sorted out and mowed over:
- Gene editing accuracy and off-target effects: CRISPR technology is still not an exact science. While it is quite precise, off-target alterations do occur. In the case of editing the genome of bacteria, we risk introducing a new bacterial strain with potentially harmful properties. This can include increasing the antibiotic resistance of some bacterial species.
- Responsible use of gene editing: Researchers and practitioners must take care to prevent the creation of bacteria with unintended abilities that could have an ecological or environmental impact.
- Difficulty validating efficacy: Bacteria that are hard to grow in a laboratory can cause issues verifying if the Cas system effectively targets the bacteria.
Conclusion
The marriage of bacteriophages and CRISPR-Cas9 technology marks a transformative leap in our fight against antibiotic resistance. This innovation offers precision, adaptability, and a potent solution to the relentless challenge posed by antibiotic-resistant bacteria. By arming phages with a CRISPR-Cas9 system, aimed to modify genes in MDR bacteria, we can prevent the post-antibiotic era.
As we forge ahead, we must remain vigilant, addressing concerns such as precision in gene editing, environmental impact, and the rigorous validation of efficacy.
What’s next for me?
Stay tuned for more content on how gene editing could revolutionize the treatment of infection and fight the possible return to the pre-antibiotic era.
