Just now, the 2020 Nobel Prize in Chemistry was announced. Emmanuel Schalponte was born in 1968 and is currently the director of the Marx Planck Institute for Biological Infection and is also a visiting professor at Umeau University in Sweden.

Just now, the 2020 Nobel Prize in Chemistry was announced. French biochemist Emmanuelle Charpentier and American biologist Jennifer Doudna won this year's award for their contribution to the new generation of gene editing technology CRISPR.

The left picture shows Sharpponte, and the right picture shows Dodner

Emmanuel Schalpponte was born in 1968. He is currently the director of the Marx Planck Institute for Biological Infection and is also a visiting professor at Umeau University in Sweden. Jennifer Dudner was born in 1964 and is currently a professor in the Department of Chemistry and Chemical Engineering at the University of California, Berkeley.

In 2011, Dudner began to cooperate with Schalponjie to develop CRISPR technology. A year later, two female biologists published a paper in the journal science and pointed out for the first time that the CRISPR-Cas9 system can "positionally" cleave DNA in in vitro experiments, significantly improving the efficiency of gene editing and laying the foundation for the development of this field. The two scientists were named the 100 most influential people in the world in 2015 by Time magazine, and have won several life science awards, including the Life Science Breakthrough Award.

CRISPR-Cas9 gene editing system is one of the most important biological discoveries of this century. In 2015, Science named it the Breakthrough of the Year; the scientists who helped the birth of this technology have also won the "Breakthrough Prize" known as the "Oscar in the Science", the "Gruber Genetics Prize" that has a profound influence in the molecular biology community, and the "Warren Alpert Prize" that commended major biomedical breakthroughs.

In the December 2014 " Global Science ", the article "Editing Genes: Faster, Accurate, Simpler" introduces the mechanism of action, development history and prospects of this technology. Although the controversy remains, there is no doubt that this emerging technology, which was named "Scientific Breakthrough of the Year" by Science magazine in 2015, is ushering in a new era.

Written by Margaret Knox

Translation by Ma Wenjing

In 1973, Stanley N. Cohen and Herbert W. Boyer found a way to change the genome of organisms and successfully inserted the DNA of frogs into bacteria. In the late 1970s, Boyer's Genetech genetically modified E. coli to carry a human gene (this gene is artificially synthesized) and finally produced insulin for the treatment of diabetes. Soon, scientists from the Salk Institute for Biological Studies in La Jolla, California, , , , CA, bred the first genetically modified mouse. These great achievements in the field of genetic engineering

These great achievements have changed the process of modern medicine. However, early genetic modification methods had two limitations: they were inaccurate and difficult to mass produce. At that time, the behavior of DNA insertion into the genome was random, and scientists could only pray for good luck, hoping that they could get a useful mutation. In 1990, researchers made leap forward. They designed proteins that can shear DNA at specific sites, breaking through the first limitation. However, every time they want to modify a piece of DNA sequence , they have to design a new protein, which is time-consuming and very hard.

Time finally came to 2012. Researchers led by Emmanuelle Charpentier of Umeå University in Sweden and Jennifer Doudna of the University of California, Berkeley reported that they have discovered a genetic mechanism in cells that allows scientists to edit genomes at an unprecedented rate, and the process is very simple. Shortly thereafter, a research team at Harvard and MIT used this technique to modify multiple loci in the cell genome at one time.

This advanced technology has accelerated the development of the genetic engineering industry and has also had a profound driving effect on genetics and medicine. Scientists can now customize genetically modified experimental animals on demand in just a few weeks, saving them from the workload and time of the previous year. At present, researchers are using this technology to explore treatments for diseases such as AIDS , Alzheimer's disease, and schizophrenia. The technology makes the genetic modification process of organisms fairly simple and inexpensive, and researchers and ethicists even begin to worry that this will create negative effects.

This technology is called CRISPR, which is the abbreviation of "clustered, regularly interspaced, short palindromic repeats" (i.e. clustered, regularly spaced short palindromic repeats). Using this sequence, bacteria can create "memory" of the virus that invades it. Scientists have been studying this strange gene sequence since Japanese scientists discovered CRISPR in the late 1980s. However, it was not until Dudna and Carpentier accidentally noticed a protein called Cas9 that CRISPR showed its huge potential as a genome editing tool.

RNA power

In 2011, Dudna and Carpentier met at a scientific conference in San Juan, Puerto Rico. They have a lot in common: their teams are all studying the mechanisms that bacteria defend against virus invasion; they have all confirmed that bacteria can remember the DNA of their viruses that have invaded their previous viruses in order to identify viruses, and when the virus invades again, they will immediately recognize the "enemy."

Shortly after that meeting, Carpentier and Dudna decided to cooperate. At that time, Carpentier's laboratory at Umeo University just discovered that streptococci seemed to use Cas9 protein to "mash" the virus that broke through its cell wall. So, Dudna also began to explore the mechanism of action of Cas9 protein in Berkeley's laboratory.

There are a series of clever things behind many scientific discoveries, and the story of CRISPR is no exception. Krzysztof Chylinski of Carpentier Laboratory and Martin Jinek of Dudna Laboratory grew up in the adjacent town, speaking the same Polish dialect. "They started chatting through Skype. The two hit it off, and then they started sharing data and discussing the idea of ​​doing experiments. That's how the project officially started." Scientists in both laboratories realized that they might be able to use the Cas9 protein for genome editing. Genome editing is a method in genetic engineering, and enzymes are the "molecular scissors" in this process that can shear DNA. This enzyme is called nuclease, which can cut off double-stranded DNA at specific sites. After DNA breaks, the cells repair the breaking site. Sometimes, some artificially imported gene fragments in the cell will insert these sites during the repair process. When Dudna and Carpentier first started working together, if scientists wanted to change or close a gene, the most advanced method was to customize an enzyme that can find and cleave specific DNA sites. In other words, every time a gene is modified, scientists have to design a new protein that specifically targets the DNA sequences they want to modify.

But Dudna and Carpentier realized that the Cas9 protein, an enzyme used by streptococci, will use RNA to guide themselves to find the target DNA. To detect the site of action, the Cas9-RNA complex will keep "bounce" on the DNA until the correct site is found. This process seems random, but it is not. Each time the Cas9 protein bounces, it is searching for the same short "signal" sequence. Cas9 attaches to DNA, detecting whether adjacent sequences match the RNA that acts as a guide. This RNA is called guide RNA (gRNA), and the Cas9 protein cleaves DNA only when the gRNA and DNA match. If this natural RNA guide system can be used, researchers will not have to build a new enzyme every time when cutting DNA sites. Genome editing may become easier, cheaper, and more effective as a result.

This transatlantic team worked together to study the Cas9 protein for several months and made a breakthrough. Dudna could still remember that moment clearly. Their laboratory is located on a tree-shaded hillside on the edge of the Berkeley campus, opposite the Greek Theater. Inek, who was still doing postdoctoral research at the time, was always experimenting with Cas9 protein. One day, he came to Dudna's office to discuss the experimental results. Faced with a question that Inek and Helinsky have been discussing, they fell into contemplation: in nature - that is, in streptococci, the Cas9 protein relies on not one, but two RNAs to guide themselves to find the right site on DNA.

What will happen if two gRNAs are integrated into one RNA strand while retaining its wizard function? If only one RNA sequence is needed, the researcher's working speed will be greatly improved. There is a delicate complementary relationship between the gRNA sequence and the target DNA sequence. Using this relationship to build a gRNA is easier than customizing a nuclease.

"Looking at the data, we suddenly got to know it - this kind of thing happens often," Dudna said. "We realized that we could actually design these RNA molecules into a gRNA. A system of a protein and a gRNA is enough to be a powerful genetic modification tool. I shuddered and thought, 'Oh my God, I'm going to go to the lab quickly, if that works...'"

They really succeeded. The result was beyond Dudna's vision (although she had high expectations). On August 17, 2012, when Dudna and Carpentier made their research on CRISPR-Cas9 public, scientists in the field immediately realized the transformative power of this technology. They all wanted to know what CRISPR-Cas9 can do, and a global competition kicked off.

Rapid commercialization

Until 2013, researchers have been trying to apply CRISPR-Cas9 to plant and animal cells—they are much more complex than bacteria. In their opinion, it was as exciting as the resurrection of Neanderthals as mammoths. At Harvard University, a team led by geneticist George Church uses CRISPR technology to change human genes, providing multiple possibilities for the treatment of diseases.

CRISPR-Cas9 quickly became a hot spot for investment. In 2013, Dudna joined hands with Church, MIT's and other researchers to jointly establish Editas Medicine, which received $43 million in venture capital to develop a new class of CRISPR-based drugs. In April 2014, CRISPR Therapeutics, which received a $25 million investment, was established in Basel, Switzerland and London, UK. Their goal is to develop CRISPR-based disease therapies. Both Aidas Pharmaceuticals and CRISPR Medical will take years to develop the corresponding therapies. However, lab suppliers are already selling CRISPR materials that can be used for animal injections to customers around the world and are starting to customize CRISPR-modified mice, rats, and rabbits for customers.

In 2014, I visited SAGE Labs in St. Louis on a wet summer day, one of the first companies to be allowed to transform rodents using Dudna’s CRISPR technology. There, I was able to see for myself how CRISPR works. SAGE Laboratory supplies experimental materials to about 20 top pharmaceutical companies, as well as numerous universities, research institutes and foundations. Horizon Discovery Group, a biotech company in Cambridge, UK, has also independently entered the research and development of CRISPR products; in September 2014, they acquired SAGE Laboratories for US$48 million. SAGE Laboratory is located in an industrial park and is built in a group of low office buildings at the end of a road.The scientists here received an online order from the lab: A lab in Sacramento, California, ordered 20 mice knocked out of the Pink1 gene to study Parkinson's disease. The newly built side building costs $2 million, which contains customized genetically modified rats, as well as other CRISPR-modified rodents. The animals live in ultra-clean, constant temperature cages that are neatly placed together, lined up from the floor to the ceiling. The staff fill out the order, select the corresponding 20 rats, gently pack them in a box, and then airlift them to California - the whole process is that simple. If someone wants to study schizophrenia or pain control, you can also order experimental animals like this.

However, if there is no animal in the warehouse that customers want to customize, the process will be different. For example, one client wanted to study the relationship between Parkinson's disease and a newly discovered suspicious gene (or a specific mutation in a gene), and when he went to the SAGE lab to order rodents, there were several options. Scientists in SAGE labs can use CRISPR technology to "turn off" the target gene and create a mutation; they can also turn off the target gene and then insert a human gene into it. From Parkinson's disease to cystic fibrosis to AIDS, many diseases are related to genetic mutations. In the past, it took scientists a year to cultivate these experimental animals with complex gene mutations. But CRISPR is different from previous genome editing techniques. Using this technology, researchers can quickly change multiple genes in cells at the same time. The time to cultivate genetically engineered animals has been reduced to several weeks. The employees of

SAGE first used a chemical kit to synthesize customized DNA and RNA that matched the DNA. They mixed RNA and Cas9 protein in a Petri dish, and a set of CRISPR tools with genome editing functions was born. Then they would spend about a week testing the function of the tool in animal cells with an instrument that resembles a scanner. This instrument is able to emit current and inject CRISPR tools into cells. The CRISPR tool entering the cell will immediately start working, shearing the DNA and inserting and deleting small amounts of genes. CRISPR is not 100% effective: in some cells, they cut DNA and create mutations, but in others it does not work at all. To see how exactly CRISPR performs, scientists collect DNA from cells, gather them together, and copy multiple copies of DNA fragments near the target site. They process and analyze the DNA and then view the analysis results displayed on the computer screen. If CRISPR successfully cuts the target site and creates a mutation, a blurry band will appear on the screen, and the more DNA CRISPR shears, the brighter the band will be.

Next, the "battlefield" was transferred to the animal laboratory on the side building. It is here that scientists create genetically modified embryos and mutated rodents. Biologist Andrew Brown bent over in front of the dissection microscope wearing surgical gloves, blue robe, overshoes and a fluffy hat. He sucked a rat embryo with the tip of the glass pipette, then walked to the other end of the room and transferred the embryo to another microscope with a robotic arm. He placed the embryo in a drop of liquid on the slide and fixed it to the countertop. Now, CRISPR is about to use its magic: he controls the joystick with his right hand and a mechanical arm pierces an empty glass needle into the embryo.

From the eyepiece of the microscope, the two pronucleus from parents in the embryo are like craters on the surface of the moon. Brown gently pushes the cells until one of the pronucleus moves next to the needle tip. He clicked on the computer mouse and a drop of liquid containing CRISPR was sprayed out from the needle and passed through the cell membrane and entered the cell. The protocore immediately expanded like a rapidly blooming flower. Brown was lucky, and a mutant cell was born. There are three technicians in the SAGE laboratory who repeat this work 300 times a day, 4 days a week.

Brown aspirates the rat embryos that have been injected into a pipette, transfers them into a Petri dish, and stores them in an incubator heated to the animal's body temperature. Finally, he needed to inject 30 to 40 modified embryos into the body of the surrogate mother. After 20 days, surrogate rats will be pregnant with 5 to 20 "children". When these "children" grow to 10 days old, scientists from SAGE laboratory will take tissue samples to test which "children" has modified genes.

"This is the most exciting time," Brown said. Among the 20 embryos, only one may be successfully transformed, and the animal that has been successfully transformed is what we call founder animal. At this point, everyone will celebrate. In our opinion, the method of making RNA and injecting embryos by scientists in SAGE laboratories seems to be very simple, and many laboratories are also using the same steps to cultivate genetically engineered animals. As SAGE CEO David Smoller said, this is a genome editing technology that can be "mass-production".

Prospects and risks

CRISPR has bravely embarked on the journey of commercialization. Researchers and businessmen are envisioning new commercial uses for this technology, and some of the ideas are even a bit arrogant. Using this technique, doctors may be able to modify abnormal chromosomes associated with Down syndrome in women in early pregnancy; breeders can reintroduce herbicide-sensitive genes into the genome of resistant weeds; we can also revive extinct species. This of course will scare some people. For example, there have been some warning headlines recently describing this technique as “a great way to play God” or “the demon in the bottle.” These articles worry that when we are eager to get rid of the malaria mosquitoes, want to cure Huntington's disease, or expect to "design" a better baby, we may also be creating a "Jurassic Park" full of harmful new genes.

Take the "Mosquito Removal Project" proposed by Harvard University researchers as an example. Todd Kuiken, a biosecurity analyst at the Woodrow Wilson International Center for Scholars, believes that defeating the malaria parasite is one thing, but to eliminate the vector of this parasite is a completely different task. If our goal is to eradicate malaria, a disease that infects 200 million people and kills 600,000 people every year, we have to be careful whether we will create 10 new troubles. "We have to think clearly, 'Are we really going to do this?' If the answer is 'yes', what systems do we have available? What safeguards are there?"

scientists are moving quickly, hoping to foresee the most likely harm of CRISPR technology and develop countermeasures. On July 17, 2014, when a Harvard team published a paper on how to use CRISPR to eliminate malaria mosquitoes, they were also calling on the public to discuss the issue, and they also pointed out the technical and regulatory dilemma of genetic modification. "CRISPR is so rapid that many people have not heard of this technology, but we are using it. It's a new phenomenon," said Jeantine Lunshof, a bioethicist at the team. Now, under the framework of Berkeley's Innovative Genomics Initiative, Dudna is forming a team specifically discussing the ethical issues of applying CRISPR.

If people's concerns about ethical issues are put out of people's enthusiasm for CRISPR, the consequences will be unimaginable. For example, in June 2014, researchers at MIT reported that they injected CRISPR directly into animals from the tail to cure adult mice with tyrosinemia (a rare liver disease). The disease is caused by a mutated enzyme. The researchers injected the mice with three gRNA sequences and Cas9 proteins, as well as the correct DNA sequences of the mutant gene. One of the mice had the correct gene inserted in every 250 liver cells. The next month, the "corrected" liver cells flourished and eventually replaced one-third of the lesion cells - enough to free the mice from the above disease.In August 2014, researchers led by Kamel Khalili, a virologist at Temple University, reported that they had used CRISPR to shear HIV in several human cell lines.

Since the 1980s, Khalilly has been fighting on the frontlines of fighting HIV/AIDS. For him, CRISPR is an absolute revolution. Although AIDS treatment has made great progress, today's drugs can only control the virus and still cannot eradicate the disease. However, using CRISPR, Khalil's team has completely eliminated the complete DNA copy of HIV from the cells and converted the infected cells into virus-free cells. And, in addition to "cleaning" cells that have been infected with the virus, CRISPR can integrate a viral sequence into uninfected cells and immunize them - as Dudna and her team observed in the original bacteria. You can call this method a "genetic vaccine." "It's the ultimate treatment, if you asked me two years ago, 'Can you accurately cut HIV in human cells?' I might say it's very difficult. But now, we've done it."