The story of CRISPR: From bacteria to the great innovation of the 21st century

In 2012, chemistry professor Jennifer A. Doudna of Berkeley University discovered a promising CRISPR-based tool that could be used to customize DNA genetics correctly. Until January 2013, they made a great step forward: Cut a piece of DNA from human cells and replace it with another piece of genetic code.

At the same time, a group of other researchers from Harvard University and the Broad Institute also independently claimed that they had successfully developed the same method. This is said to be the greatest discovery in the molecular biology of the 21st century. At the same time, this is also what will completely change the face of both human medicine and agriculture in the future.

Successive achievements

Since the successful announcement in early 2013 until now, although only for two years, researchers have conducted hundreds of experiments around the CRISPR toolkit. All aim to raise the level of human medicine and agriculture to a new level, unprecedented in human history. Some scientists have tweaked the mouse's DNA set to treat genetic diseases. Botanists have used CRISPR to modify the genes of plants and animals in hopes of creating a better food supply for humans. Even the researcher has rebuilt the full genome of a curly-haired mammoth to revive this extinct ancient species.

Portrait of biology professor Jennifer A. Doudna, one of the winners of the Breakthrough Prize by discovering a DNA custom tool based on the CRISPR system in microorganisms.

In a report published last year in the Journal of Reproductive and Endocrinology last year, Motoko Araki and Tetsuya Ishii, a group of 2 researchers from Hokkaido University, Japan predicted that doctors would be able to use CRISPR. to modify the genes of human embryos "in a very near future" . Studies around the CRISPR tool have been carried out at extremely fast speeds on a large scale.

Not only the academic world but the pharmaceutical industry also quickly paid attention to this potential tool. Many pharmaceutical companies have begun to develop and commercialize a number of drugs based on the CRISPR tool. In January of this year, pharmaceutical giant Novatis announced it could use CRISPR technology to serve cancer therapy research.

Massachusetts Institute of Technology (MIT) calls CRISPR "the greatest biological discovery of the century". Doudna's research team was awarded the Breakthrough Prize - the foundation founded by Facebook founder and CEO Mark Zuckerberg to honor those who have contributed to the development of science. Accompanying the prize is the $ 3 million grant given to the research team, almost double the Nobel prize. And this is just one of the many prestigious awards that the group is awarded.

Next, we will talk about the process of developing two CRISPR technologies as well as outline their working principles. Overall, we can say that no one really invented CRISPR. Why? You all think that the team used a way to "cut" and "paste" DNA fragments. In fact, the tool used here is "naturally available molecules" : bacteria. For millions of years, bacteria have been able to edit their own DNA and until now, they are still doing it day and night everywhere on the planet, from the deep sea floor. coming right in the human body.

Bacteria use their own ability to transform DNA as a sophisticated immune system, allowing them to learn how to identify enemies. This is the CRISPR technique which is implemented in the natural world. However, it has long been a mysterious topic waiting for scientists to answer. Although our understanding of the microbiological world is not yet complete, it is clear and promising. The study of two female professors only discovered one type of CRISPR and there are many other forms of it waiting to be discovered in the future. If all the mysteries about CRISPR are solved, people can have a more effective new tool for editing genes, and even clearing the way for other future applications.

Mysterious "string of code"

In fact, scientists have discovered CRISPR since 1987 however, they did not expect it to be a revolutionary discovery and even they did not understand what they had found. In 1987, Professor Yoshizumi Ishino and colleagues at Osaka University, Japan announced a gene sequence called iap taken from E. coli bacteria. Then, in order to better understand how genes work, the researchers sequenced the code around the original genome. The ultimate goal is to find a protein contact point to "turn on / off" iap. Ironically, instead of finding "switches" , they discovered something that was incomprehensible.

Exaggerated image of E. coli bacteria

Near the iap gene, there are 5 identical DNA segments. As we all know, DNA is made up of "bricks" called Nucleotides (Nu). In the same 5 DNA segments, each segment is made up of 29 Nu. These repeat sequences were separated by another 32 Nu on the DNA sequence, called "spacers" . Unlike repeating sequences, each "whitespace" segment has a unique sequence. This is something no biologist has ever discovered before. When Japanese researchers published the results, they also questioned this finding. They wrote: "The biological role of these spaces is unknown."

At that time, DNA decoding techniques were still quite rudimentary and the researchers could not determine whether those particular sequences were found only in E. coli or on other species. It was not until the 1990s that advanced technology contributed to speeding up the process of genetic decoding. By the end of the 90s, molecular biologists had on hand the techniques to quickly identify the genetic sequence of specimens. Even with a spoonful of seawater or a sample of soil, they can quickly determine the genetic sequence of organisms in that sample. This technique is called Metagenomes to quickly capture genetic material directly from the environment. And then, it was found that the sorting sequence discovered by Japanese scientists was not only on E. coli but also on other species.

In 2002, scientist Ruud Jansen and colleagues at Utrecht University, Netherlands, named this "repeating space gap" sequencing sequence as CRISPR - Abbreviation for: "Clustered Regularly Interspaced Short Palindromic Repeats ". At the same time, Jansen's team found that the CRISPR chain is always accompanied by a number of genomes distributed nearby. They call these genes the gene Cas, which are linked to CRISPR and capable of coding DNA-cutting enzymes. However, they still do not know why they did so and why they always go along with CRISPR.

If you have eaten yogurt or cheese, chances are you've eaten CRISPR cells

Three years later, three groups of scientists independently claimed that they had solved the mystery surrounding the CRISPR chain. Professor Eugene Koonin, who leads a research group at the US National Biotechnology Center at Bethesda, has studied the CRISPR sequence. Koonin concluded that bacteria used CRISPR as a weapon against viruses. Accordingly, bacteria do not passively respond to virus attacks, but they know how to defend effectively. Koonin hypothesizes that bacteria use Cas enzymes to grab DNA fragments from viruses. After that, they will install the captured segments into their own genetic sequence. After that, when another virus attacks, the bacteria will use this CRISPR range as a "code list" to identify the invader.

However, there have not been many other studies done to reinforce Koonin's argument. It was not until molecular biologist Rodolphe Barrangou began to test. For Barrangou, Koonin's hypothesis is not only an interesting research topic, but he also follows the orders of the famous yogurt company Danisco - a firm that has a business associated with the use of bacteria. to transform milk into yogurt.

In a validation test, Barrangou's group gave a sample of fermented yogurt with Streptococcus thermophilus virus infected with two virus standards. Initially, the virus killed a lot of bacteria, but some still survived. When the bacteria have formed antibiotics and reproduction, their descendants have been able to resist the attack of the original two species of virus. Observing the stages of the genetic transformation of bacteria over generations, Barrangou discovered that Koonin's original hypothesis was completely accurate.

Currently, Barrangou is an associate professor at the University of Northern Calirona and when asked about CRISPR, he humorously shared: "If you eat yogurt or cheese, chances are you have eaten the code-bearing cells. CRISPR ".

Cut and paste

Once CRISPR's original secret was gradually revealed, many other scientists in the industry began to be curious about it and the potential it opened. One of them was chemistry professor Jennifer A. Doudna and once she began to study more about CRISPR, she discovered that more dynamic secrets were piling up inside. Previously, Doudna was a famous professor in the industry, considered an expert in RNA (one of two DNA fragments). Basically, modern biology has seen the main work of RNA as "messenger". During cell division, RNA plays a role in transcription and transmission, which is a template for building proteins. However, Professor Doudna and colleagues discovered some other functions of RNA, such as acting as an action sensor or controlling gene activity.

In 2007, Dr. Blake Wiedenheft from the University of Montana began joining the laboratory of Professor Doudna with the aim of jointly researching the principle of operation of Cas enzymes. Although accepting this project, Doudna did not initially think of discovering any CRISPR application values. She said: "They all work to gain more knowledge, not to target a specific purpose."

During the study, the team determined the structure of the Cas enzyme and how it works in conjunction with other genetic systems. When a virus attacks the bacteria, the host cell will grab some of the virus's genetic material, cut its own DNA sequence and insert the genetic code into "spaces" . Once the CRISPR area has been filled with viral DNA, it becomes a collection that helps the bacteria identify the virus during subsequent "encounters" .

In the next attack, the bacteria will use the DNA fragment of the virus to turn the Cas enzymes into an accurate guided weapon. Then, the bacteria will copy the genetic material in the white space into a new RNA. This RNA fragment will go along with Cas enzymes floating in the cell. If it encounters the genetic material from the virus that is attacking, coinciding with the CRISPR RNA, they will tighten up. Meanwhile, Cas enzymes will cut the viral DNA into two parts to prevent it from replicating.

The video describes the CRISPR process in collaboration with Cas enzymes to help bacteria prevent virus attacks

From this unexpected discovery of the ability of bacteria, the team began to expand the ability to defend other bacteria in nature. They found that by using the database and pre-programmed, the bacteria will use enzymes to find any piece of DNA from the strange virus and stop the attack. Great, excellent, wonderfull! Professor Doudna said: "Once we have understood how microbes programmed DNA enzymes, we have an extremely unique application. God! This is really a great tool." .

In fact, this is not the first time humans have taken advantage of bacterial activity to develop biological tools. Some species of bacteria have the ability to protect themselves against attack by using molecules called limited enzymes. These enzymes will cut any piece of DNA that does not possess a protective shield. Accordingly, the bacteria will first create shields to protect its DNA and then enzymes will patrol. When any DNA fragment is "nude" , it will be processed immediately. In the 1970s, molecular biologists discovered this principle of miraculous activity and gave birth to the modern biotechnology industry like today.

Several centuries later, this kind of biological technology has improved a lot, but it still hasn't solved the important problem: Limited enzymes have no evolutionary ability to perform a single cut. exactly. That is, it only knows how to cut but cannot cut at the point that people want. As a result, the technique of using limited enzymes for cutting is only applicable in some simple requirements. However, the CRISPR-Cas system developed by Doudna's team can solve that: making extremely precise controlled cuts.

From the precise cutting technique of Professor Doudna to the excellent immune response of bacteria

From left to right, Twitter CEO Dick Costolo, Professor Emmanuelle Charpentier from Ulmea University, chemistry professor Jennifer A. Doudna from Berkeley University and actor Cameron Diaz at the Breakthrough Prize award night at the research center NASA's on November 9, 2014

To create DNA cutting tools, Doudna's team chose the CRISPR-Cas system from Streptococcus pyogenes, a bacterium that causes sore throats. This is a system that has been thoroughly studied before and also possesses cutting enzymes called Cas9 . Next, the team provided Cas9 with a piece of RNA that had the same sequence as the DNA they wanted to cut. And so the RNA segment will guide the CRISPR-Cas system to the position to be cut, "bup" , complete. In this way, the team can cut any gene segment they want. The next job is to "stitch" a new code into the available space. The team said that the tool could be used to perform any unique code from any creature they want.

Another important result of this discovery is to show an amazing ability of bacteria to help it complete the protective shield against virus attack. This is what humans have not yet discovered before and scientists call it the immune mechanism that responds . We also have innate immunity and, at the same time, we have a more advanced system that responds to immunity against pathogens by learning about them.

This system is held in some special immune cells. These cells will swallow the pathogen, then release their fragments, called antigens, and send them to other immune cells. When normal immune cells receive antigens, it duplicates cells and creates many daughter cells, randomly containing antigen. This cycle creates an army of immune cells that can quickly identify and destroy pathogens correctly. Perhaps initially, the immune system takes a certain amount of time to adapt and determine the causative factor. But once completed, this "memory" will be stored permanently.

For bacteria, they also possess this type of immune response and even more optimal. What is more optimal than people? Please, there are "lessons" on the list of pathogens that can be transmitted directly through generations. Humans cannot pass genes in antibodies to eggs or sperm to clear the way for their children. This work is only done by immune cells during human development. As a result, babies must begin to learn the pathogens from the beginning throughout their lives.

But for CRISPR, things become easier. Partly because bacteria are unicellular organisms, their DNA is evolved to fight off the virus and it is this DNA that transmits directly to the next generation through cell division. In other words, the next generation will inherit the experience of the previous generation without re-learning. So what if this ability is equipped for people?

Promises mixed with skepticism about CRISPR in the future

Artwork

Dr. Konstantin Severinov is currently working at Rutgers University and Skolkovo Institute of Science and Technology, Russia thinks that the promising applications of the CRISPR tool may become a reality but there is still so much to do. discover more. On the other hand, he said: "CRISPR's immune response function is just a rhetorical and blunt." He argues that if this mechanism helps the bacteria to genetically direct knowledge of virus enemies to generations later, over the past thousand years, the database has been immensely large. On the other hand, there are many types of viruses that have become extinct long ago, so bacteria will do it for them. It is one of the mysteries that need to be solved in the future.

However, Dr. Severinov is optimistic that: "CRISPR is a quite flexible system that can be applied in many different situations. But it should be noted that the balance of this system may be different. between species ". If scientists can gain a deeper understanding of how CRISPR works in nature, they can create more breakthrough technologies in the future.

Typically, the way to customize DNA is developed by Professor Doudna's team based on the CRISPR system. They refined a CRISPR-Cas system from the bacterium Streptococcus pyogenes and made the exact cutting process, grafting another gene into a completely new DNA sequence. A group of other researchers from Cambridge University and MIT have also done the same thing but replaced with the CRISPR-Cas system taken from another bacterium, Staphylococcus aureus. In January, scientists at the Editas did the same with the Cas9 system of Streptococcus pyogenes.

Although many other research groups are conducting a series of different tests, they are still "swimming in an ocean" of CRISPR categories. Overall, all of these studies are aimed at a common purpose of determining CRISPR's mode of operation in nature and imitating it. Once this is done successfully, the future of human life will transform in a completely spectacular way. From a new agriculture to very useful medical applications such as diagnostic tools, or cancer treatments, .

All are a promising future that started today with the discovery of CRISPR. Indeed: CRISPR is the greatest biological innovation in the 21st century.