CRISPR-Cas9 and Gene Editing: Changing our DNA

In the StoryMap The Secret of Life: How Proteins are Made, we learned that the instructions to make your body's proteins lies in your DNA, with each individual recipe to make one protein called a gene.

Moreover, we learned that if something were to damage your DNA in a specific gene, something called a mutation, then the body would not be able to correctly form that protein, possibly leading to disease. In fact, many human diseases are caused by mutations in genes, including but not limited to cancer, cystic fibrosis, Huntington's disease, and sickle cell anemia.

As a result, scientists wondered for decades: if they could somehow replace or "fix" a mutated gene in a person's DNA with a healthy, functioning copy that would allow the body to make proteins correctly, then they would be able to treat disease that way! That is, if there was a way to deactivate genes that cause disease or activate ones that can prevent disease, then this could be a potentially powerful tool to combat disease at the molecular level.

These ideas of gene therapy and genome editing became popular, but for decades, they seemed impossible. Scientists barely knew the inner workings of exactly how genes were used to make proteins at the molecular scale, so they had no clue where to even begin when it came to making targeted edits in DNA.

In the late 1990s, however, some methods of gene editing were slowly developed. The first widely-regarded technology for gene editing was the use of very small scissors called Zinc Finger Nucleases (ZFNs), which made small cuts in the DNA at user-specified locations in the DNA based on the way that the ZFN was engineered.

In 2009, ZFNs became much less widely-used, as another technology became widely popular. Around this time, scientists had discovered that Xanthomonas, a plant pathogen, infects plants by injecting a protein into plant cells that can bind to DNA and activate genes in that plant.

Scientists realized that these proteins that Xanthomonas naturally edited the genes of plants, so scientists wanted to harness these proteins to edit any genes of their own wish. And by extracting these proteins from Xanthomonas and engineering them slightly to also work on proteins, scientists had developed a new and more specific way to edit genes. These gene-editing proteins were known as TALENs.

Despite these significant advances, both ZFN and TALEN technologies are almost obsolete today due to the rise of a modern gene editing technology that has taken the world by storm. This technology is new, not even a decade old, but it is now used in thousands of laboratories, has already been used to treat human diseases, and has won two Nobel prizes.

In 1987, scientists noticed that bacteria's DNA had a very strange region. Because DNA is a recipe book to make proteins, it is usually not redundant and contains only one recipe per protein. However, in the bacterial DNA, scientists found a region with a cluster of repeats and seemingly random DNA in between. Due to the nature of these repeats, these regions of DNA were named "Clustered Regularly Interspaced Short Palindromic Repeats," or CRISPR.

When scientists first discovered these repeats, they had no reason to believe that they had anything to do with gene editing. After all, they were simply sequences of DNA in bacteria. But they had no idea what was the function of these repeats and spacers.

After years of research, scientists noticed an interesting pattern: the spacer repeats in the bacterial DNA seemed to be an exact match to virus DNA, specifically viruses that invade bacteria (called bacteriophages). As a result, scientists came up with a hypothesis: maybe these spacers were a sort of memory book so that if a virus were to attack the bacteria in the future, the bacteria would already recognize the virus's DNA and be prepared to fight back.

That is, scientists guessed that whenever a virus invaded a bacteria, the bacteria would simply refer to its "memory book" (the spacer DNA in between the repeats) to obtain information about the virus. Once doing so, the bacteria would know the exact DNA sequence of the virus. Therefore, it would recognize the invading virus and be able to attack it by cutting its DNA, almost like a bacterial immune system. And this guess turned out to be absolutely correct.

After additional research, scientists found the exact mechanism by which this worked. Once a virus invades a bacteria, the bacteria goes to its "memory book" to find the exact DNA sequence of the specific virus. Once it has this reference sequence, it attaches the sequence to a very small protein called Cas9, which acts as a scissor. Because this Cas9 scissor has the exact sequence of the virus attached to it, it can recognize any virus invaders and cut the viruses into pieces, rendering them useless.

Once scientists realized that bacteria naturally had DNA-cutting technology that could cut any DNA as long as it was given a reference sequence, they wanted to harness this technology so that they could cut any specific DNA they wanted. For example, by giving the Cas9 scissor protein a reference sequence that corresponds to a cancer gene, the Cas9 protein could then cut the cancer gene in the human body, preventing cancer proteins from being formed and potentially stopping the cancer disease. Because this DNA editing technology combines the CRISPR system of bacteria and the Cas9 protein, it is called CRISPR-Cas9.

The experiments leading to the discovery and implementation of CRISPR-Cas9 was a combined effort of hundreds of scientists around the world but was primarily spearheaded by Jennifer Doudna of the University of California at Berkeley and Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin, both of whom won the Nobel Prize in Chemistry in 2020 for their findings.

Gene editing (genome editing) is a technique that allows scientists to alter DNA, which consequently alters to protein that is formed and can thus prevent disease.

Zinc finger nucleases (ZFNs) were among the first reliable and widely-used methods of genome editing. It made use a DNA-cleavage enzyme (usually FokI), which acted as a scissor to cut DNA.

Transcription Activator-Like Effector Nucleases (TALENs) were a class of engineered proteins taken from the plant pathogen Xanthomonas that were used as genome-editing tool. TALENs were cheaper and easier to make than ZFNs, but they became less popular over time because TALEN proteins were very large, were limited to simple mutations, and did not have excellent efficiency.

CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats) is a naturally occurring mechanism in bacteria that involves the storage of viral DNA, providing the bacteria with immunity to those specific viruses through the use of the Cas9 scissor protein. The CRISPR-Cas9 system has been harnessed by scientists to edit any DNA, not just viral DNA, by providing Cas9 with specific DNA sequences.

If you would like to hear the story behind the experiments of CRISPR-Cas9 from the perspective of the actual scientists who did the experiments, below is a video from Jennifer Doudna and Emmanuelle Charpentier themselves.

While our previous discussion on DNA and genes in The Secret of Life: How Proteins are Made was centered around the idea of just producing proteins, we can see here a specific case study of how mutations in genes can cause diseases due to the production of an improperly functioning protein. And because we now know that the root cause underlying many cancers are oncogenes, we can creates medicines that specifically target these oncogenes and their protein products as a way of combating cancer.

Although CRISPR-Cas9 technology sounds promising in theory, for years, it was only done in cells grown in the lab, not in human beings. Part of the reason is due to ethical concerns (i.e. should we be allowed to edit our body's DNA?), but another reason is the lack of technology. Just because something works in the test tube does not mean it will work in practice.

However, just this year, in January 2021, CRISPR-Cas9 technology was used for the first time to treat human patients, actually successfully treating sickle cell disease and β-thalassemia. In a study published in the New England Journal of Medicine, two patients were said to be cured of beta thalassemia and sickle cell disease after their DNA was edited with CRISPR-Cas9 technology, and only time can tell how much more CRISPR-Cas9 can potentially cure.

Gene editing is definitely not without controversy. Recently, it was reported that a researcher in China genetically edited human embryos human embryos, which prompted widespread condemnation and discussions about safety and ethics. Not only is it morally questionable to give newborn babies a genetic advantage, similar to how athletes cannot take performance-enhancing drugs, but any changes made to our DNA may have lasting effects, as DNA is passed down our offspring.

Nonetheless, it cannot be disputed that the ability to alter DNA brings a plethora of potential benefits to the table, including treating genetic diseases, generating more better and more resilient crops, and even protecting endangered species. Jennifer Doudna herself says that CRISPR-Cas9 and gene editing may be "the beginning of the end of genetic diseases."

It is fascinating to realize that the answer to simple question asked about 100 years ago— how proteins are made? — has directly and indirectly led to gene editing, one of the most powerful technologies in recent scientific history.

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