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Cruise Missiles for Bacteria and Gene Therapy

December 19th, 2014: Do bacteria have immune systems?

An odd question, why would a single cell organism need a defense against infection? It turns out they do have host defenses against infection and they do have enemies that require them to have a defense department. Bacteria are subject to infection from viruses called bacteriophages. The phages can inject their DNA into a bacterium and use the bacterial systems to replicate itself and propagate the infection. Because this kills the bacterium it can be used to kill bacteria that attack us in what is called antibiosis, especially in our food supply. But with a sophisticated targeting system that is coming to light now, the bacterium can destroy the invading phage and protect itself.

With the discovery of this system comes the hope that we can exploit what has been learned about this process in fascinating ways. Like gene editing. This can be done with single base precision virtually anywhere in the genome.

But how do bacteria battle phages? Bacteriophages are tiny little viruses that look very much like the Apollo Lunar Landing Module. When they land on a bacterial cell a structure that looks like a tiny hypodermic syringe impales the bacterial cell and injects its genetic material (either DNA or RNA) into the bacterium. Within minutes the bacterial machinery for making proteins is usurped by the bacteriophage and mRNA is rapidly produced from the phage genes. New virions (virus particles) are made and escape the bacterial cell to invade and infect other bacteria.

Diagram (not to scale) of a bacteriophage invading a bacterium. Image from: Thomas Splettstoesser

The bacteria have evolved mechanisms for taking a small part of the virus’ genetic material and incorporating it into it’s own genes. The bacterial DNA at these points has a very special sequence that are known as Clustered Regularly Interspersed Palindromic Repeats (Phew!!) or CRISPR for short. RNA is transcribed from this sequence (known as crRNA) makes a complex with another short RNA and these RNAs can find the invading virus’ genetic material, bind to it (with base pair complementarity) and destroy it by bringing in a CRISPR Associated Protein (Cas). These Cas proteins are molecular scissors that chop the DNA or RNA of the virus thus disabling its ability to take over the bacterium.

Scientists quickly realized that this mechanism of targeted molecular scissors could be adapted to suit the needs of both research and therapy. So why should we care about this?

Now that we understand how the RNAs seek out a target we can use this mechanism to manipulate the genes of animals, cells, and potentially even ourselves. This can be done at a very fine level of resolution. In fact this is so accurate and can be done virtually anywhere in the genome it has become known as “gene editing”. By selecting the appropriate sequence to be edited, a special RNA called a guide RNA is designed and in combination with the bacterial nuclease Cas9 [the molecular scissors] are introduced into a cell. The scissors then cut both strands of the DNA at the precisely at the desired location, and at the same nucleotide position on both strands. This causes what is called a double strand break. The cell attempts to repair it and in so doing causes mutations at the place of the break.

Alternatively, scientists have simultaneously added a new stretch of genetic material, which in the course of the cell’s repair of the double strand break gets inserted at just that place. This has already been used to correct inherited genetic defects in animals. This is a new day for gene therapy and may well usher in real genetic treatments that have been so long sought after. In a twist of irony, this system which bacteria use to protect themselves can also be used as a completely new approach to antibiotic treatment by designing the CRISPRs to attack bacteria. To do this the guide RNA is designed to target DNA sequences of a specific strain of bacteria and then the Cas9 will chop up the bacterial genes and in turn kill the bacteria. This system is specific enough that just the strain targeted is killed, so if you only target “bad” bacteria, the “good” bacteria [like those in your gut that aid with digestion] are left alone.

One really exciting avenue is fighting drug resistant "super bugs." The CRISPR tools can be designed to just eliminate the genes that cause the bacteria to be resistant to standard antibiotics. Here the guide RNA seeks out the genes that provide antibiotic resistance to the bacteria and destroys them. This can be done such that only the gene is destroyed and leaving the bacteria alive or done such that the bacterium is killed. In either case since the genes that make the bacteria resistant to antibiotics are eliminated, then wiping out the bacteria can be done with standard antibiotics! This approach promises a new era of targeted antibiotics, where we have the molecular equivalent of guided cruise missiles making precision strikes just on the source bacteria of the infection.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

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