Deep essay: Why does CRISPR have to take the Nobel Prize?

Release date: 2017-10-10

In the spring of 2011, Professor Jennifer Doudna of the University of California at Berkeley traveled to Puerto Rico to attend the annual meeting of the American Society for Microbiology. On the second afternoon of the meeting, she went to a coffee shop with her friend John van der Oost for coffee, and it was there that she met a stylish female scientist Emmanuelle Charpentier. Jennifer probably didn't think about it. It was this time that she met and changed her entire career.

For the first time, Jennifer heard the word CRISPR in 2006. In a conversation with Professor Jill Banfield on academic cooperation, Jennifer heard something similar to Crisper. Jill didn't explain the meaning of the word. Even the word was not spelled. She just said that she wanted to seek this aspect. Research cooperation. Jill said there may be some commonality between CRISPR and RNAi, and the main research area of ​​the Jennifer group at the time was RNAi. Jennifer also very happy to promise that she will meet and discuss cooperation in the coffee shop next to the school library next week.

Jill had already been there when Jennifer arrived at the coffee shop that day. She has a notebook and several papers in front of her. After a simple chat, she began to draw a sketch on her notebook.

CRISPR Sequence Source: Jennifer Doudna

This string of diamonds and squares is CRISPR. The region represented by each diamond has the same base sequence, while the sequence of the square region is different. Jennifer immediately understood the meaning of CRISPR (clustered regular interspaced short palindromic repeats). When Jennifer asked Jill about the function of the sequence, Jill replied: Not very clear. She also said that about half of the bacteria and almost all of the archaeal genomes will exist in the genome. Obviously, if the sequence is so extensive, it must play a very important role in the maintenance of the normal function of bacteria and archaea.

Next, Jill took out the papers and excitedly summarized the work done by these papers for Jennifer. In 2005, three independent research groups found that the spacer sequence between CRISPR repeats matched the DNA fragments of some phage, and the more the number of CRISPR phage DNA fragments matched, the lower the risk of bacterial infection by phage. It is clear that CRISPR may be an important component of the immune system of bacteria and archaea to protect against phage invading. Finally, Jill gave Jennifer a look at the latest article by Kira Makarova and Eugene Koonin et al., which also puts forward the hypothesis of adaptive immune function of CRISPR.

For a long time, Jennifer has been working on RNAi function, and now it seems that CRISPR has a similar function to RNAi. For Jennifer, the temptation of the research topic of CRISPR is really too great. And she thinks that the time is also very beneficial for her: although some people have put forward the theory of CRISPR function, but no one can fully verify and analyze the complete mechanism of action, and she as a senior molecular biologist The work in this area is naturally handy.

However, Jennifer was unable to find the right person to do research in this area. The timely appearance of Blake Wiedenheft solved her problem. At the time, Blake was applying to the Jennifer team for postdoctoral research. When Jennifer asked about the research direction he was interested in, Blacke returned: Have you heard of CRISPR? There is probably no better candidate than Blake.

So the Jennifer group's CRISPR project began. Shortly after Blake joined the new lab, Danish biologist Danisco has verified that the function of CRISPR is the specific immunity of bacteria. Immediately, Stan Brouns et al. reported the key role of RNA in the CRISPR system: CRISPR is first transcribed into RNA, which is then cleaved by the enzyme into small fragments with a single repeat/space sequence that bind to viral DNA. Erik Sontheimer of Northwestern University also discovered that CRISPR RNA can induce DNA degradation through DNA-RNA pairing.

While Blake and Jennifer are excited about these new discoveries, they also realize that there are still too many fundamental issues that remain unresolved. First, they need to analyze the process of integration of viral DNA into CRISPR sequences and CRISPR transcription and RNA fragment cleavage. Second, they also need to analyze the process by which CRISPR RNA induces viral DNA degradation. So they extended their eyes to the CRISPR related gene cas.

Cas gene source: Jennifer Doudna

The discovery of the cas gene is an important contribution to understanding the CRISPR function. In 2002, Jansen first used the term CRISPR in his published article to analyze the characteristics of CRISPR sequences by bioinformatics calculations and identified four CRISPR associated systems (cas) cas 1-4. The cas genes are all adjacent to the CRISPR locus, suggesting that there may be a functional correlation between the two. Moreover, the Cas protein has both a helicase and nuclease domains, indicating that they may be involved in DNA metabolism and gene regulation processes.

In order to study the function of Cas protein, Blake first obtained a large number of Cas1 proteins through genetic engineering techniques. After obtaining the Cas1 protein, he revealed the function of the protein through a series of experiments, and found that the Cas1 protein can cleave the DNA fragment to enable the viral DNA sequence to be inserted into the CRISPR sequence. At this time, Rachel Haurwitz also joined the study and participated in the study of Cas 6 to determine the function of Cas 6 to cut the transcribed long-chain CRISPR RNA. They then analyzed more and more Cas protein functions, but these proteins are very similar to the protein functions of Cas1 or Cas6.

In 2011, many old members of the group also joined the CRISPR team. At this time, the interest of Blake and Jennifer has gradually shifted from the Cas protein that cuts CRISPR DNA/RNA to the Cas protein that cuts the viral DNA sequence. Their collaboration with the John group found that the process of cutting viral DNA is extremely complex, requiring multiple Cas proteins to target and cleave viral DNA: First, CRISPR RNA will form approximately 10 Cas proteins that can be targeted for cleavage. A complex of viral DNA sequences followed by Cas3 will cleave the target DNA sequence. Later, Jennifer worked with other groups to resolve the structure of the recognition shear sequence complex and determined the important role of base pairing in the recognition process. A laboratory in Lithuania also determined how the Cas3 protein is cut: Cas3 is not cut once, but is cut into hundreds of DNA fragments.

As the research progresses, researchers are gradually recognizing the extremely high diversity of the CRISPR system. In general, the CRISPR system is divided into two categories, six groups, and 19 subgroups. The study of the Jennifer group before 2011 only focused on the first category, but she knew very little about how the second type of CRISPR cuts DNA. At this point she entered the bottleneck of research.

II

In early 2011, Jennifer met Emmanuelle Charpentier at the annual meeting of the American Society for Microbiology. When John introduced her to Emma, ​​she immediately remembered her student's wonderful report on tracrRNA at the meeting of Emma at Wageningen. Emma has been interested in CRISPR since the beginning of 2000. She collaborated with Max's J?rgVogel to sequence small RNAs in S. pyogenes and found that the tracrRNA content in the bacteria is extremely abundant. Through bioinformatics analysis, they predicted that the gene sequence encoding the RNA was adjacent to the CRISPR site and speculated that it might affect the function of CRISPR. A series of subsequent experiments showed that the system has three components: CRISPR RNA, Csn1 protein, and tracrRNA.

Left: Jennifer Doudna; Right: Emmanuelle Charpentier

However, the mechanism of the system is not clear to her. Cooperation with Jennifer is a more sensible choice. Jennifer was very excited about the cooperation. She had not been involved in the research of the second type of CRISPR system, and this cooperation provided a new direction for her work. She quickly designated the group's post-Martin Jinek, as well as the master exchange student Michael Hauer from Germany as a participant, and the Emma group's doctoral student Krzysztof Chylinski also joined the research project.

In fact, Jennifer realized the important role of Csn1 in the first conversation with Emma. The Csn1 protein is later known as Cas9. As early as 2007, Rodolphe and Philippe reported that Cas9 can resist the infection of Streptococcus thermophilus. Josiane and Sylvain and others also found that the inactivation of Cas9 protein affects the DNA DNA shearing process. Emma's team found that mutations in the cas9 gene affect the production of CRISPR RNA and cause damage to the entire immune process. Further research by Virginijus Siksnys et al. found that Cas9 is the only Cas protein that plays an important role in the CRISPR system of Streptococcus thermophilus. There is increasing evidence that Cas9 plays a very important role in viral DNA cleavage in the second type of CRISPR system.

However, how to analyze the mechanism of the CRISPR system? Obviously, the first step is to figure out the function of Cas9. Similar to the previous work, the first thing to do is to extract and purify the Cas9 protein. Krzysztof sent the artificial chromosome containing the cas9 gene to the Jennifer group, who completed the task. Since Martin is busy looking for a faculty member at this time, he can only instruct Michael to complete the purification of the protein. But when Michael mixed Cas9, CRISPR, and the corresponding viral DNA, he found that Cas9 couldn't cut the viral DNA, and Michael's deadline for communication in the lab was up. He had to return to Germany to graduate.

On the eve of Michael's return to China, Martin finally found a faculty at the University of Zurich and succeeded Michael. He and Krzysztof successfully found the reason why Michael's experiment failed: the lack of tracrRNA. After day and night work, the whole process of the Cas9 shearing process gradually emerges: First, Cas9 needs to bind to the viral DNA, and open the double strand of DNA to enable CRISPR RNA to base pair with a single strand of DNA. The two nuclease functional modules of Cas9 simultaneously cleave two DNA single strands to break the DNA. With the transformation of CRISPR RNA, the system can cleave almost all viral DNA sequences that can be paired with CRISPRRNA.

So can Cas9 cut other DNA sequences besides the viral DNA sequence? If possible, CRISPR will be a new gene editing tool. The field of gene editing has broad application prospects, but the existing nucleases based on TALE and zinc finger proteins have serious limitations. The main problem is that the efficiency of pairing proteins with DNA sequences is too high. The Cas9 protein cleavage only requires the pairing of CRISPR RNA and viral DNA sequences to complete the cleavage. If Cas9 can be used as a gene editing tool to cut other types of DNA sequences, then Cas9 will revolutionize the field of gene editing.

To simplify the gene editing system, Martin linked CRISPR RNA to TracrRNA to form a chimeric RNA strand. Next, in order to verify the ability of the system other than the cleavage of viral DNA, he selected five different sequences consisting of 20 bases in green fluorescent protein (GFP) and obtained a chimeric RNA capable of pairing with it. After mixing the RNA with Cas9 and GFP DNA, as expected, all of the GFP DNA was perfectly cleaved at the set site.

On June 8, 2012, Jennifer and Emma submitted papers to the Science Journal, and the article was received only twenty days later. Even though they are very clear that this article will have a huge impact on the field of genetic editing, they never expected that CRISPR would set off such a stormy wave.

III

Jennifer and Emma's article laid the foundation for the application of CRISPR as a gene editing tool, but this article only verified the ability of the system to shear free GFP DNA, and whether the CRISPR system can cleave DNA in cells becomes another urgent need for verification. The problem. So Martin and Jennifer didn't dare to stop for a moment, and immediately began research in that direction. First, Martin transferred the DNA sequences encoding Cas9 and the guide RNA into two plasmids and introduced the plasmid into the cells. By transcription, Cas9 protein and guide RNA were generated, and the DNA was cleaved inside the cells. Since studies have shown that ZFN can successfully edit the CLTA gene in the human embryonic kidney cell line HEK293, they chose the same gene, the same cells, in order to compare the pros and cons of the two gene editing methods.

However, long before the publication of Jennifer and Emma's article, many people have already predicted the huge potential of CRISPR, including Zhang Feng and Geroge Church. Born in 1982, Zhang Feng is the youngest core member of the Harvard-Broad Institute of Harvard and MIT. During his Ph.D. at Stanford University, he helped create optogenetic tools that have had a profound impact on research in the field of neuroscience. After graduating, he returned to Harvard University for genetic editing and became the first scientist to use TALE to control mammalian genes.

In 2011, a visiting scholar went to Rhodes to study the report, and the subject of the report was about adaptive immune-related CRISPR in bacteria. He was sitting in the back row of the lecture hall, and his thoughts were not focused on the content of the report, but the Greek word, which sounded a bit strange, aroused his interest. He has never heard of CRISPR before, but he is getting more and more excited when searching for relevant information online. A few days after listening to the report, he went to Miami to attend an academic conference, but he did not have the heart to listen to the conference report, and he was always in the hotel room to browse the relevant information of CRISPR.

Fortunately, there were not many articles published at the time. Since it was previously discovered that CRISPR can counter the virus that affects the manufacture of yogurt, most of the applied research at that time was concentrated in the yogurt manufacturing industry. But Zhang Feng has a bold idea: he wants to apply CRISPR to human cells. Although the study of the genetic editing tool TALE continues to be less risky for him, it is clear that Zhang Feng is not a fear of failure.

Left: Zhang Feng; Right: George Church

After returning to the office, Zhang Feng told his students about his thoughts, and he immediately understood why Zhang Feng was so excited about CRISPR. Previously, the TALE they used required a cumbersome process of protein synthesis, and often after the protein was synthesized, it was found that the protein could not be targeted to the target DNA sequence. However, unlike CRISPR, only RNA is required to recognize the target DNA sequence. If synthetic protein is to build a skyscraper, the synthesis of RNA is as simple as building a two-story building.

However, Zhang Feng and Qi Le did not start researching bacteria like Jennifer and Martin, but experimented directly with human and mouse cells. If they want to bring innovation to the medical industry, they must prove the potential of CRISPR in these cells. The two started a crazy job. At the time, they had two goals: to prove that CRISPR was able to edit the animal cell genome, and that the edited genome could produce the desired function. They also chose the gene encoding the GFP protein as the target for gene editing. The fewer cells that produce green fluorescence after editing, the more cells that CRISPR has successfully edited.

Zhang Feng and Cong Le did not realize the existence of competition at the time. In June 2012, Jennifer and Emma's article was launched, which validated the ability of CRISPR to cleave free DNA and demonstrated the potential of CRISPR as a gene editing tool. But Zhang Feng is not frustrated by the appearance of this article, because there is a huge difference between cutting free DNA and editing the genome of animal cells. Finally, they completed the work of the subject a few months after the Jennifer article was published. In January 2013, Zhang Feng’s article was published in Science. He also didn't realize that his former mentor, George Church of Harvard University, was doing the same job at the time. A similar work by George was also published in the same issue of Science.

For many scientists, the protection of intellectual property is as important as publishing an article. On May 25, 2012, Jennifer et al. filed a patent application to protect CRISPR technology. Seven months later, on December 12, Zhang Feng also filed a patent application. Zhang Feng was the first to obtain a patent license in April 2014 due to the application for accelerated patent review. In January 2016, Jennifer's University of California requested a patent for the original patent of the Broad Institute and 11 other patents, which led to the famous CRISPR patent war. In the end, the patent dispute ended with the Broad Institute.

But I think for many people, the most important thing is not who is the first to publish an article, who is the first to get a patent, and finally which people get the Nobel Prize, most people are concerned that the CRISPR technology will be for scientific research, medical care, Even the impact of mass life. At some level, this is why CRISPR must win the Nobel Prize. We will cover this in more detail in the second half.

In order to clarify that CRISPR is a Nobel Prize-winning result, the author has introduced the basic concepts and detailed development process of CRISPR technology in the first half of the article. For details, see: Deep essay: Why does CRISPR have to take the Nobel Prize? (on). The second half of the article will focus on the application of CRISPR technology in various fields, and a brief introduction to relevant listed companies.

IV

Plant genome editing

At the beginning of 2016, DuPont announced that the news of developing a new type of waxy corn did not attract much attention. What everyone doesn't know is that DuPont's technology "CRISPR" to cultivate waxy corn is quietly changing the entire breeding industry. The company uses CRISPR technology to knock out the glycoside gene wx1, which encodes starch synthase in corn, thereby reducing the amylose content of corn and increasing the amylopectin content, thereby increasing the viscosity of corn.

In the billions of years since the birth of life, the evolution of life has followed Darwin's theory of species evolution: the advantages of survival, competition and reproduction of offspring through random genetic mutation. But from the beginning of farming civilization, humans have tried to change the outside world and selectively cultivate better animals and plants. Early farmers used new varieties or hybrids to obtain new varieties, but this is similar to the natural evolutionary process, and also relies on random mutations in DNA. So the improvement process is very slow, although it has been very small after thousands of years of progress.

Mendel's work at the beginning of the twentieth century laid the scientific foundation for plant breeding and provided a predictable framework for breeding. After World War II, with the development of biotechnology, new breeding methods have emerged, such as the use of mutagenic agents such as ethyl methanesulfonate or dimethyl sulfate, or the use of ionizing radiation or transposons to induce DNA mutations. New crop traits. Modern breeding is a further step, and the genome of plants can be directly modified by molecular biology. For example, genetic engineering is used to introduce specific genes into plants to form transgenic crops.

However, genetic recombination technology has its own limitations, such as difficulty in accurately mapping to the genome, or knocking out or knocking down genes. So how do you accurately locate the genome for gene editing? In 1996, the Srinivasan Chandrasegaran team at Johns Hopkins University discovered that the Zinc finger protein with genomic localization function and Fokl endonuclease were linked together to form a tool for precise mapping and cutting of DNA. . However, this technology has many problems, its zinc finger protein for genome localization is poorly programmable, and the design and synthesis process is very long. And California's Sangamo company firmly controls the patent for this technology, thus greatly limiting the development and application of the technology.

After more than a decade, another more instructive genome editing technique, TALEN, has enabled scientists to edit genomes quickly and accurately. TALEN was named Method of the Year by Nature Method in 2011, but TALEN was only under the turbulent waves of CRISPR for only a year or two.

CRISPR is used in painting by Gregory Allen

Thanks to the advent of CRISPR technology, scientists are able to manipulate the genome of crops with unprecedented precision. In fact, CRISPR technology not only enables breeding experts to more easily obtain the expected crop traits, but also can be used to improve the disease resistance of a variety of crops. In the short span of several years, the technology was used to edit the wheat genome to protect against bacterial blight and to make corn, soybeans and potatoes resistant to herbicides. In 2014, scientists at the Chinese Academy of Sciences used CRISPR and TALEN technology to simultaneously modify six gene copies of the wheat Mlo gene to make it resistant to powdery mildew. More and more crops are gaining income and CRISPR technology, making it more resilient.

At the same time, CRISPR's advancement in plant biology research is enormous. Scientists have long explored the function of genes in crops by observing natural mutations in nature or by artificially inducing random mutations. However, CRISPR can explore the function of genes by introducing mutations into genes, destroying the coding region of genes, and inducing dysfunctional proteins in a faster and more efficient way. In addition, CRISPR can also be used to target miRNAs to activate or inhibit the expression of specific plant genes, or to knock in, replace, and even use dCas9 to regulate plant gene transcription.

V

Animal genome editing

In addition to food crops, CRISPR technology can have a profound impact on the livestock industry, with very small genome modifications that greatly increase the meat production of farmed animals. And similar to the application in crops, the use of CRISPR technology can easily modify multiple genes in animals at the same time. For example, Chinese scientists use CRISPR to simultaneously modify the myostatin gene MSTN and the growth factor gene FGF5, which can control hair growth, while improving Goat meat production and hair quality.

Although the animals edited by CRISPR can promote the development of animal husbandry, the field of experimental animal research can best reflect the unlimited potential of CRISPR technology. Whether used for pathological research or new drug evaluation, experimental animals are extremely important for modern medicine. The most basic of experimental animal research, the most important thing is to obtain a reliable animal model, in order to simulate the external manifestations and intrinsic pathogenesis of human pathogenesis.

Since the beginning of the last century, mice have become the most commonly used mammalian model in biomedical research. Today there are more than 30,000 mouse strains for research on a range of diseases ranging from cancer to cardiovascular disease and blindness. The emergence of CRISPR technology provides an efficient and fast technical means for the establishment of mouse models. Not only for almost all mouse strains, but also for greatly reducing the time required, and at the same time being relatively inexpensive, the cost is only about one-tenth that of traditional genetic tools.

Although mouse animal models are widely used, they still have a number of limitations. For diseases such as cystic fibrosis, Parkinson's disease, and Alzheimer's disease, they usually fail to exhibit characteristic symptoms of the disease, and atypical reactions may also occur in the evaluation of the efficacy. This makes it extremely difficult for laboratory research to transform into clinical trial research. The emergence of CRISPR technology has made the establishment of non-human primate models more efficient.

Although foreign genes could be transferred into the monkey genome by viruses more than a decade ago, scientists were unable to edit the monkey genome before the emergence of CRISPR technology. At the beginning of 2014, Huang Xingxu of the Institute of Model Animals of Nanjing University injected CRISPR into single-cell embryos, and introduced the Ppar-γ and Rag1 combinatorial mutations to precisely modify the genome of cynomolgus monkeys, thus obtaining the world's first gene knock. Except monkeys.

CRISPR-Cas9 Horse Baby; by MichaelCammer

In addition to mice and monkeys, pigs have become an important animal model due to the emergence of CRISPR technology. The anatomical structure of pigs is similar to humans. The organ size is similar to that of humans. It has a short reproductive cycle and a large number of litters. It can also be used as an animal model, but more importantly, pig organs may become human organ transplants. Important source. In fact, this has been the dream of some scientists for a long time, but it is out of reach due to technical limitations. Even organ transplants between humans can cause severe immune rejection, not to mention transplants between different species. Moreover, the presence of porcine endogenous retroviruses (PERVs) is also a major safety hazard.

The emergence of CRISPR technology has made us a big step toward the pursuit of pig organs as a source of human organ transplantation. Previous techniques have largely evaded immune rejection by transferring certain genes from the pig genome, but gene editing techniques, including CRISPR, can directly knock out genes that cause immune responses, or directly knock out PERVs.

In 2015, George Church of Harvard University and his student Yang Lan jointly established eGenesis, hoping to use CRISPR technology to achieve the grand goal of human organ transplantation in pigs. In the same year, they achieved the feat of using CRISPR to simultaneously knock out 62 sites of the PERVs gene in the pig genome. Then they completed another achievement: getting piglets without the PERVs gene. They first knocked out the PERVs gene of the pig embryonic connective tissue genome without triggering apoptosis. Then, the nucleus was transferred into the pig egg cells by cloning technology, and then the embryo was transplanted into the pig uterus and the pig was successfully produced. kid.

Although there is still a long way to go in the future of pig organ applications, the unlimited potential of CRISPR has allowed us to change earlier to see this day. Because of Yang Lan’s contribution to the medical field, she was selected by the World Economic Forum as the “Global Youth Leader of 2017”.

In addition to medical-related applications, the brain-studded GeorgeChurch is also working on a well-known project: the resurrection of mammoths. Two mammoth specimens that died between two 20,000 to 60,000 years ago provided the possibility for whole-genome sequencing. Genomic analysis can be used to obtain genomic changes between mammoths and existing elephants, and to discover 1668 genetic differences in proteins associated with body temperature perception, skin and hair development, and adipose tissue formation. In 2015, the George group successfully replaced 14 of the modern elephants with the genes of the mammoth using CRISPR technology, but replacing all the genes will undoubtedly make a very large project, and the modified elephant cells may not be able to Clone and develop into an embryo. (GeorgeChurch published a book a few years ago to explain his grand goal in detail. This book has a great brain and includes how to synthesize human "chiral isomers" and other research projects).

Resurrection mammoth By NBC

In addition to these applications, scientists are also using CRISPR technology to control genetic processes and modify the genetic information of their offspring. This technology is called Genedrive. In the sexual reproduction of diploid organisms, the offspring obtain a set of chromosome copies from both parents, which means that the parental gene (except selfish gene) has a 50% chance of being passed on to the offspring. But using Genedrive technology can change the way genetic information is transmitted.

Leading the project is Kevin Esvelt of the GeorgeChurch (and his) group. At the heart of this technology is the knock-in of genes, using CRISPR technology to precisely cut specific sites and insert a new sequence. The inserted sequence contains information for generating the CRISPR gene editing system, so it can automatically copy itself to another chromosome, so that all individuals of the progeny contain information that can encode the CRISPR system.

If the inserted sequence contains not only the CRISPR information but also other information, the information can also spread rapidly in the children. For example, Genedrive is used to insert a Plasmodium resistance gene into the mosquito genome. In theory, all mosquitoes in the region will carry the Plasmodium resistance gene after a period of time. This will be a major advance in the prevention of malaria. However, many scientists have not stopped here. They envisage the insertion of a female sterility-related gene in the mosquito genome, so that the gene spreads like a virus in some mosquito populations, causing the mosquito population to rapidly become extinct in this region.

This can be a terrible technology, and scientists in the laboratory should do their best to prevent genetically modified mosquitoes from spreading to the outside world. In the process of release, it is difficult to predict how much the impact will spread, and if the mosquitoes in some areas are quickly extinct, although some scientists claim that they will not have serious impact, personally, the ecological impact will be difficult to predict. The ecosystem is not as simple as some models predict.

In addition, the most important question is how to prevent the malicious use of the technology? The design of Genedrive is not very difficult. If someone inserts some malignant genes into the mosquito genome, the technology will immediately become Gene Bomb. How to use this technology safely will be a very big problem.

VI

Disease treatment

Preclinical experimental studies of various animal models have demonstrated the enormous potential of CRISPR in preclinical animal models and provide an important tool for disease research and drug discovery. But can CRISPR technology be used more directly to treat diseases?

In less than a year after Zhang Feng and GeorgeChurch Labs demonstrated the feasibility of editing human cells in 2013, the Li Jinsong team of the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences used the same genetic editing tools to verify their treatment of genetic diseases. potential. They selected a mouse model of cataract genetic disease in which the model mice carrying the dominant mutation of the Crygc gene were able to produce denatured lens proteins and turbidity led to cataracts. Results CRISPR was able to find and repair mutations from approximately 2.8 billion base pairs in the mouse genome.

The researchers designed a targeting RNA directed against the mutant Crygc gene and injected the targeting RNA and Cas9 directly into the fertilized egg of the heterozygote, and found that one-third of the newborn mice had cured cataract symptoms. Moreover, the cured mice can pass the Crygc gene repaired by the CRISPR-Cas9 system to the next generation through germ cells. In the following years, scientists used CRISPR to cure muscle atrophy and liver metabolism-related diseases in mice, and proved the potential of this technology to treat sickle cell anemia, hemophilia, cystic fibrosis, severe combined immunodeficiency and other major diseases. . Whether it is a nucleotide mutation, a deletion/increase, or even a chromosomal abnormality, CRISPR seems to be competent.

All of these studies provide preliminary validation of the safety and efficacy of CRISPR technology directly applied to the treatment of human genetic diseases. Of course, the potential of CRISPR is far more than just for the treatment of genetic diseases. Scientists are also trying to use genetic editing to prevent human cells from being infected with the virus. In fact, the first genetic editing clinical trial before this was to treat HIV infection. In addition to the field of infectious diseases, cancer is also one of the areas of application of CRISPR technology.

Gene Editor By Gloria Pizzilli

Although genetic editing is a very powerful tool, it is not easy to turn the results of animal experiments into clinical research. The ups and downs of gene therapy in the past few decades have reminded us that medical progress is far more difficult than we think. The first question scientists face is the choice of target cells, somatic cells or embryonic cells?

By modifying single-cell embryos, the genome of all cells will be altered after development, and the genomes inherited by their offspring will also be altered. Animal model experiments also demonstrate the feasibility of this strategy. But this method faces this serious ethical problem. So the somatic cells became the first choice for most scientists. The genome modification of somatic cells cannot be passed on to offspring and thus reduces ethical problems. However, genome editing of somatic cells is much more complicated than that of germ cell theory, so we must solve the new problems brought by the selection of somatic cells for genome editing.

The biggest problem is the delivery of the drug. Different diseases affect different parts of the body. For example, Huntington's disease mainly affects brain neurons, while sickle cell anemia affects red blood cells, and cystic fibrosis mainly affects the lungs. Choosing a more accessible internal circulation system is a relatively easy direction. There are two types of treatments for circulatory systems: in vivo gene editing and in vitro gene editing (ex vivo). Relatively speaking, gene editing in vitro is easier and better for quality control. Last year, the Lu ur team of West China Hospital of Sichuan University took the lead in launching the world's first CRISPR human clinical trial, knocking out the star molecule PD-1 on the surface of T cells. What the team used was the way in vitro gene editing.

But not all scientists have the same ideas as above. First, choose gene editing using somatic cells.

In March 2015, five scholars published a joint article in the Nature of Don't edit the human germline (do not edit human germ cells, it sounds a bit like three-body monitors reply to Ye Wenjie's three sentences do not answer), calling on researchers to be cautious Edit the germ cell genome using gene editing tools. But only a month later, Huang Jun from Sun Yat-sen University published an article on the group, which reported the use of CRISPR technology to edit 86 inactive human embryos in order to modify the HBB gene that can cause thalassemia. Although the results of the experiment were not ideal, the article caused great controversy internationally due to ethical issues. Many people worry that if CRISPR is used to modify the human embryonic genome to prevent genetic diseases, then the technology will be difficult to avoid and applied to modify non-medical related genetic problems. Despite this, the Yellow Army was selected by Nature magazine as the top ten scientific figures of the year.

Editor's Embryo: Who is playing the role of God? (Genesis Mural by Michelangelo)

由于该文章所引发的巨大争议，同年美国、英国和中国在华盛顿联合组织了人类基因组编辑国际峰会，对人类基因组编辑的安全问题、伦理问题和政府监管进行了讨论。在这之后，胚胎基因编辑的伦理争议似乎开始变得没那么激烈，2016年瑞典和英国成为了中国之外的，允许胚胎进行基因编辑的另外两个国家。

由于技术越来越成熟，该领域的研究和文章也越来越多。截止目前共有8篇人类胚胎编辑的文章发表，而其中5篇是在过去两个月内发表的。两周前(9月22日) 黄军就的另一篇文章上线，同样是修饰HBB基因用于治疗地中海贫血，但这一次使用的是克隆胚胎细胞，而且利用的是不具有剪切功能的CRISPR系统(且携带胞苷脱氨酶)进行碱基编辑，以修饰点突变。

毫无疑问运用CRISPR进行人胚胎基因组编辑能够对人类疾病的预防产生巨大的影响，虽然现在的研究重点主要集中于体细胞基因组编辑，用以治疗疾病，但随着技术的不断进步，CRISPR编辑生殖细胞的潜力会被进一步挖掘，伦理问题也可能因此得到比较好的解决。

VII

CRISPR相关生物技术公司

嗜热链球菌能够将乳糖转化为乳酸，所以常用于奶制品行业。丹麦的Danisco公司首先证明了嗜热链球菌含有的CRISPR序列的功能是细菌的适应性免疫，用以抵御噬菌体入侵（见：深度长文：为什么CRISPR必须拿诺奖？（上））。2011年杜邦公司收购了Danisco，并开始研究如何利用CRISPR抵抗噬菌体感染的嗜热链球菌，更好的制造酸奶和奶酪。同年，Jennifer Doudna还在研究第一类CRISPR系统的时候，她就参与创立了Caribou Biosciences，希望运用CRISPR技术来简化病毒检测的过程。

Emmanuelle Charpentier在2012年也逐渐有了创立公司的想法。在Science文章上线的五个月后，她便与当时还在赛诺菲任高管的老友Rodger Novak以及另一位风险投资家的老友Shaun Foy讨论CRISPR的商业潜力。一个月后Novak决定辞职共同创立一家新公司。之后三人开始积极寻找合作伙伴。

在与Jennifer交流之后他们计划联合George Church和张锋共同创立这家公司，以期简化之后可能存在的专利问题。但不幸的是，由于各种已知和未知的原因之后的商谈极其不顺利。在张锋和Church文章发表的一年半以后，CRISPR技术已经越来越成熟，资本强烈期望介入，成立公司的需求也越来越迫切。

CRISPR专利听证会；来源： Science

但在知识产权、学术信誉、地理因素、媒体报道、诺贝尔奖、商业回报等一系列因素的考量下，对CRISPR技术做出巨大贡献的四个人不仅没有团结一致，反而开始分崩离析、各自为政。Jennifer和Emma二人组的关系也不像从前那么单纯，变得越来越微妙。不仅个人的利益掺杂其中，加州大学、布罗德研究所、哈佛大学、麻省理工、维也纳大学这几个学术机构的利益之争也让局面越来越复杂。

在此之后，Emmanuelle Charpentier, Rodger Novak, Shaun Foy, 以及Chad Cowan共同成立了CRISPR Therapeutics。张锋，George Church, Jennifer Doudna共同成立了Editas Medicine。Erik Sontheimer, Luciano Marraffini, Derrick Rossi和Rodolphe Barrangou共同成立了Intellia Therapeutics。而之后入局的其他公司则需要向以上这几家公司和布罗德研究所支付高昂的专利授权费。

在2014年关键专利授权给张锋之后一个月，Jennifer离开了Editas，加入Intellia。

VIII

写在最后

我想现在已经很难找到一个没有听说过CRISPR这个名词的生物专业学生。短短几年内CRISPR技术已经为基础科研领域，和包括医疗健康以及农业等与大众生活更相关的领域的发展产生了巨大的推动作用。在科学的发展过程中，偶尔会有重大的进展出现，比如相对论，比如DNA双螺旋，比如PCR技术，CRISPR虽然可能不能与以上的突破比肩，但其对科学发展的影响是毋庸置疑的。

Jennifer和Emma的工作为CRISPR作为基因编辑工具的诞生提供了基础，而张锋和Church两人同时证明了CRISPR编辑哺乳动物细胞的巨大潜力。毫无疑问，这些人对于CRISPR技术的诞生与发展做出了重要贡献。但如果没有Jennifer，如果没有Emma，或者没有Church，没有张锋，CRISPR技术就不会出现了吗？显然不是(参见附录的时间线)。

CRISPR能拿诺奖的实力争议很小，但谁最后能够拿到诺奖却是个很大的谜团。科学界并不像人们想象的那样单纯，在利益和荣誉面前多数会变的不理性。在关于CRISPR的战争中，有人获得了金钱，有人获得了荣耀，有人开心，但也有人失落。

如果说CRISPR技术推动了科学的进展，使我们更加憧憬未来的美好生活，那么关于CRISPR技术的利益之战和荣誉之争，却更让我们理解了人的本性。

CRISPR Me by Michele Tragakiss

附录：CRISPR时间线

CRISPR的发现与其功能

1987年，日本大阪大学石野良纯第一次发现了CRISPR序列。

1993年，西班牙阿利坎特大学Francisco Mojica第一个确定现今被称为CRISPR的位点。

2000年，Francisco Mojica发现并报告了之前发现的该差别重复序列存在某些共同的特征（他在与RuudJansen的通信中使用过CRISPR这一名词，后者在2002年在文章中正式使用这一名词）。

2005年，Francisco Mojica发现该序列与噬菌体基因组中的某些片段向匹配，并由此推测CRISPR属于适应性免疫系统。另一课题组也在同期独立报道了类似的研究(Pourcelet al., 2005)。

Cas9与PAM的发现

2005年5月，法国国家农业研究院（INRA）Alexander Bolotin, 在研究嗜热链球菌的过程中发现了一个与众不同的CRISPR位点(Bolotin et al., 2005)，该CRISPR系统缺乏之前熟知的cas蛋白基因，而还有其他未报到过的新cas蛋白基因，其中之一正是编码后来被命名为Cas9蛋白的cas基因。而且他发现，与病毒DNA匹配的间隔序列一端均含有一个相同序列PAM(protospacer adjacent motif)，PAM是CRISPR目标序列识别所必须的。

适应性免疫假说

2006年3月，美国国家生物技术信息中心Eugene Koonin通过计算分析直系同源蛋白，并提出了CRISPR级联为基于插入噬菌体同源DNA到间隔序列的细菌免疫系统的假设，摒弃了之前关于Cas蛋白作为DNA修复系统的假设(Makarovaet al., 2006)。

适应性免疫功能的实验验证

2007年3月，Danisco公司Philippe Horvath试图研究广泛应用于奶制品工业的嗜热链球菌对于噬菌体的反应。Horvath及其同事通过实验发现CRISPR系统确实属于适应性免疫：将新的噬菌体DNA片段整合入CRISPR，并用以抵抗该类噬菌体的下一次攻击(Barrangou et al., 2007)。他们还发现Cas9可能是使入侵噬菌体失活过程中唯一必须的蛋白。

间隔序列被转录为向导RNA

2008年8月，荷兰瓦赫宁根大学John van der Oost开始逐渐解析出CRISPR-Cas系统干扰噬菌体入侵的过程。Johnvan der Oost 及其同事发现来源于噬菌体的间隔序列能够转录生产小RNA，成为CRISPR RNAs (crRNAs)，能够引导Cas蛋白靶向目标基因(Brouns et al., 2008)。

CRISPR如何作用于DNA靶标

2008年12月，美国西北大学Luciano Marraffini 和Erik Sontheimer发现系统作用的靶标是DNA而非RNA (Marraffini and Sontheimer, 2008)。由于人们一直以来都认为CRISPR和RNAi具有类似的机制，这一发现让研究人员感到意外。Marraffini和Sontheimer 表示该系统如果能够转移到非细菌系统中，将可能开发成为强大的工具(需要注意的是有的CRISPR系统可以靶向RNA (Hale etal., 2009)。

Cas9 剪切目标DNA

2010年12月，加拿大拉瓦尔大学Sylvain Moineau及其同事发现可以靶向DNA并在精确位点（PAM上游三个核苷酸）引起双链断裂(Garneau et al., 2010)。他们同时确证Cas9是CRISPR-Cas9体系剪切过程所需的唯一蛋白、由单个蛋白（此处为Cas9蛋白）和crRNAs介导的干扰过程是第二类CRISPR系统的独有特征。

Cas9 系统中tracrRNA的发现

2011年3月， Emmanuelle Charpentier课题组完成CRISPR-Cas9系统介导干扰过程的机制解析的最后一块拼图。他们通过包含CRISPR-Cas9系统的产脓链球菌测序发现除了crRNA，尚存在第二个RNA，称为tracrRNA(trans-activating CRISPR RNA) (Deltcheva et al., 2011)。该RNA与crRNA形成双链，并介导Cas9靶向DNA靶标。

CRISPR 系统可以在异种生物中起作用

2011年7月，立陶宛维尔纽斯大学,Virginijus Siksnys及其同事克隆了嗜热球杆菌（含有第二类CRISPR系统）的整个CRISPR-Cas位点，并在大肠杆菌（不含有第二类CRISPR系统）中表达，并发现其能够气功质粒抗性(Sapranauskaset al., 2011)。以上实验表明具有独立的功能，而且第二类系统所需的组分都已发现。

Cas9介导的剪切过程解析

2012年9月，立陶宛维尔纽斯大学Virginijus Siksnys及其同事利用以上在大肠杆菌中表达的系统确证了Cas9的作用机制(Gasiunas et al., 2012)。他们确定了PAM的作用以及剪切位点，通过点突变发现RuvC结构域能够剪切非互补链，而HNH结构域能够剪切互补连。同时表明crRNA序列最少只需要20个核苷酸，更重要的是他们发现通过更改crRNA序列可以引导Cas9靶向对应的DNA位点。

2012年6月，美国加州大学伯克利分校Emmanuelle Charpentier 和Jennifer Doudna与Virginijus几乎同时报道了相同的发现(Jinek et al., 2012，该文章提交时间晚于Gasiunas等人文章)。但他们发现crRNA与tracrRNA能够融合形成一条RNA，简化了该系统。同时他们报道了运用该系统对GFPDNA进行剪切。

应用CRISPR-Cas9 进行基因组编辑的验证

2013年1月，哈佛-麻省理工布罗德研究所张锋和哈佛大学George Church在同期的科学杂志报道了运用CRISPR-Cas9系统成功进行真核细胞内的基因组编辑。他们的研究表明CRISPR-Cas9系统能够剪切人类以及老鼠细胞内基因组的多个位点，并能引导同源重组修复。

来源：医药魔方数据（微信号iyiyaomofang）

16' PVC Coated Gloves

16' Pvc Coated Gloves,40Cm Red Pvc Gloves,Anti Chemical Pvc Gloves,16Inch Pvc Working Gloves

JINAN SHANDE SECURITY TECHNOLOGY CO., LTD , https://www.sdsxlb.com