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Manipulating the forces of nature with gene editing This 2005 study explains the discovery of CRISPR-cas9, the tool used in gene editing. It has been used in research on agriculture, cancer, infectious disease, genetic disorders, and more.

When Dr. Ido from Jurassic Park said, “Your scientists were so preoccupied with whether they could, they didn’t stop to think if they should,” he was referring to reviving the dinosaurs. On a grander scale, he was talking about the lines people were willing to cross for the sake of science. 

Manipulating the forces of nature may seem dangerous but it’s something we’ve been doing for centuries. Early farmers were “mad” scientists with a history of “editing” nature. Before we even knew what genes were, farmers mixed and matched plant traits to breed food that would be more flavorful and longer-lasting. If it weren’t for these field experiments, tomatoes would still be bitter berries and corn would still be grass. Messing with plants doesn’t seem too harmful, so when is it going too far? This question was revisited with the invention of CRISPR-CAS9.

CRISPR-CAS9 is a tool that has transformed the field of gene editing. Gene editing first starts with DNA, the building blocks of life. Different gene combinations can determine our height, our hair color, and more. DNA can also influence our health. A single mistake in our genetic code could create mutations that have long-term consequences. Sickle cell disease, for example, is a genetic disorder that occurs when there is a mutation with the HBB gene. This gene is important because it gives instructions for making a protein called beta-globin. When beta-globin is smashed together with another protein called alpha-globin, it makes a bigger protein in red blood cells that help carry oxygen throughout the body. A change in the instruction or mutation means the proper proteins will not be made.

Genes are arranged in patterns of only 4 molecules, represented by “A,” “T,” “C,” and “G.” In this case, one of the instructions requires the “A” molecule, but in sickle cell anemia the “A” is replaced with a “T” molecule. The letter swap creates a mutation where it prevents proteins from folding. The dysfunctional proteins mean red blood cells can’t access tissue, which needs the oxygen carried by red blood cells to properly function.

This is an illustration representing a gene. The “letters” here represent specific molecules that attach to each other. CNX OpenStax / CC BY

In theory, there is a simple solution. Switch the T back with the letter A. In practice, this is way more complicated as DNA is like a super-long recipe with specific instructions. Its recipe can stretch from a few thousand to over two million “letters”. The concept of gene editing relies on replacing a defective gene with a healthy one. It turns out, it’s very hard to find a specific location to place a gene. It’s like finding a single word in a 1000 page book (without using Control+F!)  As a result, scientists resigned themselves to sticking the gene somewhere random and hoping for the best. This randomization meant a low probability of success and possibly sticking the gene in the wrong place. This could lead to even more mutations leading to mixed-up instructions and worse outcomes like cancer and death. This is where CRISPR-CAS9 comes in.

In the late 1990s, Dr. Francisco Mojica was only a graduate student studying salt-loving microbes called Haloferax mediterranei in the seawater of Spain. His original goal was to study why these organisms adapted to extremely salty environments. Since these single-celled organisms have a simpler genetic make-up than humans with small amounts of DNA compared to the millions of DNA sequences in humans, they looked at their genes to see if their love for salt was a genetic adaptation. What surprised him was an unusual place where a DNA sequence was repeated several times with spaces in between each repeat. It wasn’t until 2002 when his team discovered the same thing in bacteria.

CRISPR-modified rice” by pennstatenews is licensed with CC BY-NC-ND 2.0.

What was the purpose of the repeated spacers? How does having it in the DNA benefit bacterial survival? In his 2005 paper, Dr. Mojica looked at other single-celled bacteria like E. coli. His research team used a technique to make billions of copies of the bacterial DNA. Then, they used a computer program to find the repeated and spaced out DNA sequence. They found the same repeated and spaced out DNA segments in other bacteria. These repeats were also found in viruses that invaded bacteria. This suggested that these segments, which they called CRISPR, may have evolved as part of an immune response against invaders.

CRISPR isn’t the only one on the job. In 2005, Dr. Bolotin found CRISPR’s right-hand man to be none other than the cas9 gene. She was studying bacteria that lived in dairy products when she came across CRISPR. But there was something off about this particular CRISPR segment as it had a cluster of foreign cas genes including a large protein called cas9. Her team also found that the spaces in the CRISPR segment all ended in the same pattern. This 2-6 base pair fragment of DNA called PAM was found to be 3-4 spaces away from where the Cas9 protein had cut up DNA. This protein is referred to as “molecular scissors” because of this cutting ability. They also observed that bacteria with the most PAM sequences were also the most resistant to viruses. Dr. Bolotin and her colleagues concluded that PAM is used to identify viral targets that need to be cut up by Cas9. They also reached the same conclusion as Dr. Mojica’s — Cas9 must be part of the bacterial immune system.

How genes get inserted with CRISPR and the PAM sequence. “File:GRNA-Cas9.png” by marius walter is licensed with CC BY-SA 4.0.

The idea of CRISPR as an immune response was strengthened by findings of the first examples of CRISPR in action: yogurt. Yogurt has “good” bacteria called probiotics important for our gut health. But maintaining the bacteria was a big issue as the majority died when they came into contact with a virus. Viruses have only one goal in mind: to hijack a cell’s DNA and pump out more copies of itself. Bacteria that did survive viral invasions were analyzed by researchers at a yogurt company where they found new spaced-out DNA repeats identical to the virus. When cas genes were inserted into the bacteria, they eventually became resistant to the virus. However, if the new DNA segment was somehow removed, the bacteria would instantly die. 

Scientists agreed that CRISPR helps to protect bacteria from invaders. It’s possible that somehow CRISPR allowed bacteria to easily identify or have an immune memory of the virus. It’s similar to the bacteria creating a most-wanted poster of the RNA strand of the virus (humans use DNA, viruses use RNA as their genetic material). The cas9 protein is given the wanted poster where it patrols around DNA looking for a match. If it does locate a copy of the viral strand, the cas9 protein will cut up the DNA segment like a pair of scissors.

At the time, Dr. Mojica believed his work on CRISPR would only be used to help understand the immune system of bacteria. It wasn’t until 2012 when several researchers would discover how helpful CRISPR-CAS9 would be as a gene-editing technology when it was successfully used on human and mouse cells. What makes CRISPR-CAS9 revolutionary is that it gives precise, targeted changes to DNA. 

CRISPR-CAS9 acts as a search function in Microsoft Word where it easily searches through pages of text on Microsoft Word to find the incorrect or defective letter and deletes it to type in a new one. Going back to the sickle cell anemia example, with CRISPR -CAS9, it’s easier and safer to cut up the wrong letter in DNAr and add the correct one to a specific location. There are endless possibilities to use CRISPR-CAS9. Currently, CRISPR-CAS9 therapies are underway for curing genetic diseases.Other applications include crops being edited to be more nutritious, designing cancer treatments, and possibly bringing the dead back to life! There have also been ethically controversial measures including editing human embryos and plans to create designer babies. Where do we draw the line for gene editing? Scientists are still debating this question but for now, it’s clear that we’ve entered a whole new relationship with nature.

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Study Information

Original study: Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements

Study published on: February 2005

Study author(s): Francisco J.M. Mojica, Chc)sar Díez-Villaseñor, Jesús García-Martínez & Elena Soria

The study was done at: Universidad de Alicante & Génétique Microbienne, Institut National de la Recherche Agronomique

The study was funded by:

Raw data availability:

Featured image credit: "CRISPR Cas9" by National Institutes of Health (NIH) is marked under CC PDM 1.0. To view the terms, visit https://creativecommons.org/publicdomain/mark/1.0/