A New Application of CRISPR

By Lily Zimmerman

Bacteria: tiny organisms that do everything from causing sickness to carrying out important internal bodily functions. We often view bacteria as gross or harmful creatures, but in reality, they are absolutely essential to our survival. Bacteria, particularly the billions that populate the gut, are incredibly important; they help regulate the immune system, assist with digestion, and even play a role in the central nervous system, which impacts our mental health. However, the gut microbiome is also a sensitive ecosystem and can be disturbed by many factors such as diet, stress, or antibiotics, leading to serious imbalances, which can cause issues with any of the functions listed above. Issues with gut bacteria can lead to many detrimental health impacts for the organism they populate. A new study entitled, “Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome,” published in Cell Reports and conducted by researchers at University of San Francisco, describes a new method by which we may be able to correct issues with the gut microbiome. Researchers hope to use CRISPR, a new technology for genetic manipulation, to find a mechanism for the genetic modification of bacteria. Genetic modification could be a powerful tool for combating the issues that arise in the gut microbiome.

Though there are current methods used to correct gut microbiome imbalances and improve microbiome diversity such as fecal transplants and probiotic pills, these methods don’t always have the desired effect. Researchers hope that genetic modification of the microbiome might be a more reliable and effective method for correcting imbalances. When treating other diseases, genetic modification could also be a way to target only the problematic bacteria and avoid harming good bacteria strains, unlike treatments such as antibiotics that target all bacteria. 

There have been very few attempts to genetically modify gut bacteria and little research on the practicality of it. However, the CRISPR CAS-9 system is a relatively new mechanism for gene modification that is a huge focus point for current research. The CRISPR CAS system is made up of two key molecules: the Cas 9 enzyme and a guide RNA. The Cas 9 enzyme is what cuts the strand of DNA at a specific location, allowing a new DNA sequence to be added or a segment of DNA to be removed. The guide RNA allows the Cas 9 enzyme to target certain spots on the DNA strand. It contains a segment called the scaffold that is engineered to bind to a specific point on the DNA and also attract the Cas 9 enzyme, directing it to the correct portion of DNA. Once the Cas 9 enzyme is attached, it can induce the desired genetic changes on the DNA. Researchers hope to use this technology as a basis for the manipulation of E. coli bacteria. Researchers wanted to determine the effectiveness of a mechanism they believe is capable of genetically modifying bacteria in the mammalian GI tract. In order for the desired components to be inserted, they needed a way to enter the E coli. They hoped to use the M13 gene to do this, as M13 is a virus that can target, infect, and insert strands of DNA into E. coli. It is a type of bacteriophage, a virus that is able to infect bacteria. Researchers suspected that it could be a means to deliver the components required for genetic manipulation, if the necessary material is inserted into the M13. Through a series of experiments, they tested M13’s ability to insert these genes into E. coli within the mouse gut microbiome and then determined if successful genetic modification had occurred.

The first part of the experiment involved determining whether or not M13 could insert a desired gene into E coli bacteria found in mice. Researchers infected a group of mice with E. coli bacteria and gave them water containing M13; however, an antibiotic resistance gene had been inserted into the M13 virus. The antibiotic resistance gene would allow researchers to determine if the M13 had successfully transferred genetic components into the E coli: if the transfer was successful, then the E. coli would survive treatment of antibiotics. This is exactly what happened. When the E. coli infected mice were treated with the M13 containing resistance, E. coli remained in their system even after they had been treated with antibiotics, meaning M13 had successfully transferred antibiotic resistance and therefore other inserted material. 

After they determined that they could insert genetic material into the E. coli, researchers had to see if they could actually induce the desired genetic changes into the bacteria. They inserted the CRISPR CAS system along with guide RNA into the M13, programming it to induce deletions at certain points. The team then exposed E. coli in vitro to the M13 and found that this mechanism was capable of causing the desired genetic modifications. After DNA sequencing, the majority of the E. coli did contain the desired deletions in DNA. However, they also found  that the E. coli was able to repair some of the induced deletions, making the genetic manipulation slightly less effective. 

Finally, researchers had to determine whether or not they could induce these same changes in the actual gut microbiome, rather than in vitro. They gave E. coli infected mice with water containing the M13 gene and tested E. coli collected in the mouse stool. After performing genetic sequencing on the E. coli, they determined that the desired deletions had occurred; however, there was a range in the size of the deletions, meaning again that the E. coli was able to repair some of the induced deletions on its own. The deletions present do however, show that the inserted CRISPR-Cas system is capable of causing genetic alterations in the E. coli. 

The series of experiments showed that M13 containing a CRISPR system is able to cause genetic alterations in E. coli. However, the most important takeaway from this experiment is that it demonstrates the capability of genetically modifying bacteria that exist within the gut. E. coli is a common species of gut bacteria, and modification tools for E. coli specifically are useful for some cases, but our gut contains an extremely diverse array of bacteria, all of which are very important. Dr. Peter Turnbaugh, communications lead for the project, described in an interview that E. coli is a relatively easy bacteria to genetically edit, and serves as a simpler and easier model system for the future modification of other types of bacteria. Thus, modification of E. coli is a good starting point when it comes to manipulating the gut microbiome, but each bacteria will require a different mechanism. The long term goal is to be able to induce genetic modifications in any strain of gut bacteria, which Turnbaugh says is a long way down the road. Researchers will focus next on finding mechanisms that are similar to M13 and are capable of modifying other bacterial species. 

Eventually, researchers hope to develop a technique to alter the makeup of the gut microbiome to correct imbalances present in humans. Another component researchers have to figure out is how to apply this research, which has so far only involved animals, to humans too. Turnbaugh describes how current attempts have only focused on inducing modifications in mice and in vitro, so a significant amount of further trials involving humans is required before it may be a practical treatment. It is likely that, like many other health issues, the future of gut microbiome treatment will be dominated by CRISPR based genetic manipulation. 

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