Bacterial enzymes have played an important role in modern biotechnology and molecular biology through their ability to manipulate complicated molecules like DNA. In the late 1960’s and early 1970’s, scientists studying how bacteria defend themselves from viruses identified proteins called restriction enzymes that cut specific sequences of viral DNA and prevent viruses from infecting and killing bacteria. This discovery was the basis for the Nobel Prize in medicine in 1978. Shortly afterward in the 1980’s, a molecule from a different type of bacteria that lives in hot springs was used as a tool to amplify pieces of DNA (a commonly used scientific method known as polymerase chain reaction or PCR), leading to another Nobel Prize in chemistry in 1993. These bacterial enzymes have become standard tools for anyone who wants to cut and paste sequences of DNA for therapeutic or engineering purposes. Many of the medicines and foods that we take for granted today were developed using these methods. For instance, recombinant insulin, cancer drugs, and enzyme replacement therapy drugs have been generated using these tools; as well as transgenic crop plants such as BT cotton, “golden rice“, and the brand new “arctic apples” just approved for use in the US.
A recent discovery that holds similar promise is the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mechanism. This turns out to be yet another mechanism that bacteria use to defend themselves from invading viruses (Figure 1). Unlike restriction enzymes, which recognize and cut specific, short sequences of DNA, CRISPR uses a DNA-cutting protein (known as Cas9) plus short sequences of RNA (known as guide RNAs) to target and cut up viral DNA and prevent infection. The CRISPR system has been adapted by scientists to create an easy way to target the Cas9 protein to specific spots in the human genome, the DNA that acts as a set of instructions for the cells in our bodies.
You might ask: “Why would I want to send a protein to damage a specific place in my genome?” The answer is that for many diseases, changes in the DNA sequence of specific genes (known as mutations) change the instructions that a cell receives, the changes can lead to the disease. Until now we have not had reliable tools to fix these “typos” except in very specific circumstances in the lab. Developing a reliable way to fix them would allow us to address the causes of many diseases at the source. CRISPR is not the first tool available to target specific sequences of DNA (see zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)), but it is the first to rely on RNA to guide the protein, rather than having to generate a brand new protein for every sequence to be targeted. This makes CRISPR flexible and quick to use compared to previous methods. While producing and optimizing new proteins is a time-consuming task, RNA molecules can be chemically synthesized overnight.
An exciting example of how this technology can be used is repairing genes in individuals with inherited diseases. By selectively damaging DNA at the site of a disease-causing gene, cells will try to fix the damage by any means necessary. One process that they use is similar to copying and pasting to fix a typo in a text document. Since we have two copies of most genes, the cell can use the undamaged copy as a template to fix the damaged copy (Figure 2). Scientists have already used CRISPR to stimulate this process and repair mutations in mouse models of several diseases. In one study, scientists treated mice that carry a mutation the leading to the human disease hereditary tyrosinemia with the CRISPR system. The mutation that causes this disease leads to liver damage, weight loss and death. Mice treated with CRISPR to repair the disease gene initially showed a small number (0.4%) of liver cells that had been repaired a few days after treatment, but the liver is special: it has a very high capacity for regeneration and these newly-healthy cells grew and replaced the unrepaired diseased cells and after a month, they made up about one-third of the liver in the treated mice. These mice showed improvement in their ability to maintain body weight compared to animals that did not receive the full CRISPR treatment.
We still have lots of work to do before experiments like this can be attempted in people: no one takes the idea of cutting parts of the genome lightly, since damaging the wrong gene can lead to cancer or cell death. But these initial experiments show that the method can work. Currently, both academic scientists and biotech companies are pursuing this method to develop therapeutics for Cancer, Cystic Fibrosis, HIV, Huntington’s Disease, and Sickle Cell Disease. So while potential therapies from this method lie years in the future, this new tool taken from bacteria has opened up the possibility that we will be able to fix the “typos” that cause genetic diseases that were previously beyond our reach.