In the spring of 2011, Jennifer Doudna of the University of California, Berkeley, travelled to Puerto Rico to attend the annual meeting of the American Microbiology Society. On the afternoon of the second day of the meeting, she went to a coffee shop with her friend John van der Oost, where she met Emmanuelle Charpentier, a stylish female scientist. Jennifer probably wouldn’t have thought that it was this encounter that changed her entire career.
Jennifer first heard the word CRISPR in 2006. In a conversation with Prof. Jill Banfield on a discussion of academic cooperation, Jennifer heard something that sounds like Crisper. Jill did not explain the meaning of the word. Even the spelling of the word was not mentioned. She just said that she wanted to seek this aspect. Research cooperation.
Jill said that there may be some commonalities between CRISPR and RNAi, and that the main research area of the Jennifer team at that time was RNAi. Jennifer also very readily agreed that she would meet and discuss the cooperation next week in the cafe next to the school library.
When Jennifer arrived at the coffee shop that day, Jill already waited there. There was a notebook and several papers in front of her. After a simple chat, she began sketching on her laptop.
CRISPR Sequences. Source: Jennifer Doudna
The string of diamonds and squares is the CRISPR. Each diamond represents a region with the same base sequence, while the square region has a different sequence. Jennifer immediately understood the meaning of CRISPR (clustered ordered 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 genome will have this sequence. Obviously, if the sequence is so extensive, it must also play a very important role in the maintenance of the normal functions of bacteria and archaea.
Jill then took out those 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 is completely matched with the DNA fragments of certain bacteriophage, and the more CRISPR matches the phage DNA fragments, the lower the risk of bacterial infection by bacteriophage.
It is clear that CRISPR may be an important component of the immune system of bacteria and archaea to protect against phage invasion. Finally, Jill gave Jennifer a look at the latest article published by Kira Makarova and Eugene Koonin, who also put forward the hypothesis of CRISPR adaptive immunity.
For a long time, Jennifer has been engaged in the research of RNAi function, and now it seems that CRISPR has similar functions with RNAi. For Jennifer, the temptation of CRISPR is really too great. And she thinks the time is also very beneficial to her: Although some people have put forward the theory about the function of CRISPR, but no one can fully verify and resolve the complete mechanism of action, and she is doing as a senior molecular biologist The work in this area is naturally handy.
However, Jennifer finds it difficult to find suitable people to do research in this area. The timely emergence of Blake Wiedenheft solved her problems. At the time, Blake was applying for a postdoctoral research mission to the Jennifer Group. When Jennifer asked about the research direction he was interested in, Blacke replied: Have you heard of CRISPR? There is probably no better candidate than Blake.
So the Jennifer CRISPR project started. Shortly after Blake joined the new laboratory, Danisco, a Danish biological company, has verified that the function of CRISPR is precisely the specific immunity of bacteria. Immediately, Stan Brouns et al. reported on the key role of RNA in the CRISPR system: CRISPR was first transcribed as RNA and then enzymatically cleaved into small fragments with a single repeat/spacer sequence and then bound to viral DNA. And Erik Sontheimer of Northwestern University also discovered that CRISPR RNA can induce DNA degradation through DNA-RNA pairing.
While Blake and Jennifer were excited about these new discoveries, they also realized that there are still too many fundamental issues that have yet to be resolved. First they need to analyze the integration of viral DNA into CRISPR sequences and CRISPR transcription and RNA fragmentation. Second, they also need to analyze the process of CRISPR RNA-induced viral DNA degradation. So they expanded their eyes to the CRISPR-related gene cas.
Cas gene Source: Jennifer Doudna
The discovery of the cas gene has an important contribution to understanding the function of CRISPR. In 2002, Jansen used the term CRISPR for the first time in a published article. He analyzed the characteristics of CRISPR sequences and identified four CRISPR-associated genes (cas) cas1-4 by bioinformatics calculations.
All cas genes are adjacent to the CRISPR locus, suggesting that there may be a functional correlation between the two. Furthermore, the Cas protein has both a helicase and a nuclease domain, indicating that they may be involved in DNA metabolism and gene regulation processes.
In order to study the function of the 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 DNA fragments so that the viral DNA sequence can be embedded in 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 cleave the transcribed long-chain CRISPR RNA. Afterwards they analyzed more and more functions of the Cas protein, but these proteins are very similar to Cas1 or Cas6 proteins.
By 2011, many old members of the group also joined the CRISPR team. At this point Blake and Jennifer’s interest has gradually shifted from the Cas protein that cuts CRISPR DNA/RNA to the Cas protein that cleaves the viral DNA sequence. Their collaborative research with the John group found that the process of cutting viral DNA is extremely complex and requires multiple Cas proteins to target and cleave viral DNA: First, CRISPR RNA will be formed with approximately 10 Cas proteins to target the desired cleavage A complex of viral DNA sequences, followed by Cas3, cleaves the target DNA sequence.
Afterwards, Jennifer collaborated with other groups to analyze the structure of the complex that recognizes the cleavage sequence and determined the important role of base pairing in the recognition process. A laboratory in Lithuania also determined the cleavage of the Cas3 protein: Cas3 is not cut once, but it is cut into hundreds of DNA fragments.
With the deepening of research, researchers have gradually realized the extremely high diversity of the CRISPR system. In general, the CRISPR system is divided into two major groups, six groups and 19 subgroups. Before 2011, the Jennifer group focused on the first category. She knew little about how the second type of CRISPR cuts DNA. At this point she entered the bottleneck period of the study.
In early 2011, Jennifer met Emmanuelle Charpentier at the annual meeting of the American Microbiological Society. When John introduced Emma to her, she immediately remembered her students’ mention of Emma’s wonderful report on tracrRNA at the Wageningen meeting.
Emma has been very interested in CRISPR since the early 2000s. She collaborated with Jörg Vogel of Maps Institute to sequence small RNAs in Streptococcus pyogenes and found that the amount of tracrRNA in the bacteria is extremely abundant. By bioinformatics analysis they predicted that the gene sequence encoding the RNA was close to the CRISPR site and speculated that it might affect CRISPR function. 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, she did not know the mechanism of the system, and working with Jennifer would be her wise choice. Jennifer was very excited about the cooperation. She hadn’t been involved in the second type of CRISPR system research before, and this cooperation has provided her work with a new direction. She quickly appointed Martin Jinek from the group, and Michael Hauer, a master exchange student from Germany, as a participant. Dr. Krzysztof Chylinski from the Emma group also joined the project.
In fact, Jennifer already 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 et al. reported that Cas9 can resist S. thermophilus virus infection. The work of Josiane and Sylvain et al. also found that the inactivation of Cas9 protein affects the shearing process of viral DNA.
Emma’s group found that mutations in the cas9 gene affect CRISPR RNA production and result in impaired immunity. The research of Virginijus Siksnys et al. went even further. They found that Cas9 is the only Cas protein that plays an important role in the CRISPR system of Streptococcus thermophilus. More and more evidence shows that Cas9 plays a very important role in viral DNA shearing in the second CRISPR system.
However, how do you analyze the mechanism of action of this CRISPR system? Obviously, the first step is to clarify the function of Cas9. Similar to previous work, the first thing to do is to extract and purify Cas9 protein. Krzysztof sent the artificial chromosomes containing the cas9 gene to the Jennifer group and they were assigned to complete the task.
Since Martin is currently busy looking for teaching positions, he can only direct Michael to complete the purification of the protein. But when Michael mixed Cas9, CRISPR RNA and the corresponding viral DNA, he found that Cas9 could not cut the viral DNA. At this time, Michael’s deadline for the exchange in the laboratory had also arrived. He had only returned to Germany to prepare for graduation.
On the eve of Michael’s return to the country, Martin finally found a position at the University of Zurich and succeeded Michael. He and Krzysztof successfully found the reason for the failure of the Michael experiment: the absence of tracrRNA. After a long day of work, the entire Cas9 shear process gradually surfaced: First, Cas9 needs to bind to the viral DNA, and open the double strand of DNA so that CRISPR RNA can base pair with a single strand of DNA, and the pairing will succeed. The two nuclease functional modules of Cas9 were induced to cleave both DNA single strands and break the DNA. With the transformation of CRISPR RNA, the system can cut almost all viral DNA sequences that can pair with CRISPR RNA.
So can Cas9 cut other DNA sequences other than viral DNA sequences? If possible, CRISPR may become 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 pairing of protein and DNA sequences is too inefficient.
The Cas9 protein cleavage requires only 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 to excise viral DNA, he chose five different sequences of 20 bases in green fluorescent protein (GFP) and obtained chimeric RNAs that can be paired with it. After mixing RNA with Cas9 and GFP DNA, as expected, all GFP DNA was perfectly cut at the set position.
On June 8, 2012, Jennifer and Emma submitted the paper 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 gene editing, they never expected CRISPR to set off such a storm.
Jennifer and Emma’s article lays the groundwork for the use of CRISPR as a gene editing tool, but this article only validated the system’s ability to cleave free GFP DNA, and whether the CRISPR system can cleave DNA within cells has become another imperative for validation. The problem. So Martin and Jennifer dared not stop for a moment and immediately began the study in that direction.
First, Martin converted the Cas9- and guide-RNA-encoding DNA sequences into two plasmids and introduced the plasmids into the cells. By transcription, Cas9 protein and guide RNA were generated and the DNA was cleaved intracellularly. 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 and the same cell in order to compare the pros and cons of the two gene editing methods.
However, long before the articles of Jennifer and Emma were published, many people had already predicted the potential of CRISPR, including Zhang Feng and Geroge Church. Born in 1982, Zhang Feng is the youngest core member of the Broad Institute of Harvard and M.I.T. During his Ph.D. at Stanford University he helped create optogenetics tools that have had a profound effect on research in the field of neuroscience. After graduation, he returned to Harvard University for genetic editing research and became the first scientist to use TALE to control mammalian genes.
In 2011, a visiting scholar went to the Rhodes Institute for a report, and the subject of the report was about CRISPR-associated CRISPR in bacteria. He sat in the back row of the lecture hall, his thoughts did not focus on the content of the report, but CRISPR sounded a little strange term has aroused his interest. He had never heard of CRISPR before. He became more and more excited when he searched for relevant information on the Internet. A few days after hearing the report, he went to Miami to attend an academic conference. However, he did not completely listen to the conference report and was still in the hotel room to browse CRISPR related information.
Fortunately, there were not many published articles at the time. Since CRISPR was previously found to be able to fight viruses that affect yogurt production, most of the applied research at the time was focused on the yogurt manufacturing industry. But Zhang Feng has a bold idea: He wants to apply CRISPR to human cells. Although continuing to engage in the study of gene editing tool TALE is less risky for him, it is clear that Zhang Feng is not a person who is afraid of failure.
Left: Zhang Feng Right: George Church
After the return, Zhang Feng told his students Cong Le, and Cong immediately understood why Zhang Feng was so excited about CRISPR. Previously, the TALEs they used required tedious protein synthesis, and often the protein was synthesized and found to be unable to target the target DNA sequence. Unlike CRISPR, however, 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 Cong did not start with bacteria like Jennifer and Martin. Instead, they 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 crazy work. At the time, they had two goals: to prove that CRISPR can edit the animal cell genome, and that the edited genome can produce the intended 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 will successfully edit.
Zhang Feng and Cong did not realize the existence of competition at that time. In June 2012, an article by Jennifer and Emma went online. This article validated the ability of CRISPR to cleave free DNA and demonstrated the potential of CRISPR as a gene editing tool. However, Zhang Feng did not feel frustrated because of the appearance of this article, because there is a huge difference between cutting free DNA and editing the genome in animal cells. Finally, they completed the work of the topic a few months after Jennifer’s article was published. In January 2013, Zhang Feng’s article was published in Science. He also did not realize that his former teacher, George Church of Harvard University, was doing the same job. George’s similar work was also published in the same issue of Science.
For many scientists, the protection of intellectual property rights is as important as publishing articles. On May 25, 2012, Jennifer and others submitted a patent application for the protection of CRISPR technology. On December 12, seven months later, Zhang Feng submitted a patent application. Thanks to the application for accelerating the examination of patents, Zhang Feng obtained the patent authorization for the first time in April 2014. In January 2016, the University of California where Jennifer was located requested a patent intervention on Broadfield’s earliest patent and 11 other patents, triggering the famous CRISPR patent war.
But I think for many people, the most important thing is not who predates the article, who first gets the patent, and which few people finally get the Nobel Prize. Most people are concerned about whether CRISPR technology will be used for scientific research or medical treatment. Even the impact of mass life.
In early 2016, DuPont announced that it wanted to develop a new type of waxy corn and did not attract much attention. What we do not know is that DuPont’s CRISPR technology for raising waxy corn is quietly changing the entire breeding industry. The company uses CRISPR technology to knock out the enamel gene wx1, which codes for starch synthase, in corn, thereby reducing the content of amylose in corn and increasing the content of amylopectin, thereby increasing the viscosity of corn.
In the billions of years since the birth of life, the evolutionary process of life has followed Darwin’s theory of species evolution: the advantages of surviving, competing, and multiplying through random genetic mutations. However, from the beginning of farming civilization, humans have tried to change the external world and selectively cultivate more excellent animals and plants. Early farmers obtained new varieties through random breeds or using crosses, but this is similar to the natural evolution process and also relies on random mutations in DNA. So the process of improvement is very slow. Although it has gone through thousands of years, it is very small.
The work of Mendel in the early 20th 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 mutamethyl ethylsulfate, dimethyl sulfate and other mutagens, or the use of ionizing radiation or transposables to induce the formation of DNA mutations. New crop traits. Modern breeding science is even more advanced. It can directly use molecular biology methods to transform the plant’s genome. For example, genetically modified crops are formed by introducing specific genes into plants through gene recombination.
However, gene recombination technology has its own limitations, such as difficulty in accurately locating the genome or knocking out or knocking down the gene. So how to accurately locate the genome for gene editing? In 1996, the team of Srinivasan Chandrasegaran at Johns Hopkins University discovered that the Zinc finger protein and Fokl endonuclease, which have genomic location function, were linked to form a tool that can precisely position and cut DNA. . However, there are many problems with this technology. The zinc finger proteins used for genome location are poor in programmability, and the design and synthesis process is very long. Moreover, California’s Sangamo company firmly controls the patent of this technology, which greatly limits the development and application of this technology.
Ten years later another TALEN, a more programmatic genome editing technology, was created to allow scientists to quickly and accurately edit genomes. TALEN was named the Method of the Year of the year by the Nature Method in 2011. However, the TALEN was only for a year or two, and it was submerged under the torrent of CRISPR.
CRISPR is applied to corn breeding (painting by Gregory Allen)
With the advent of CRISPR technology, scientists can manipulate the genome of crops with unprecedented precision. In fact, CRISPR technology not only makes it easier for breeding experts to obtain the desired crop traits, but it can also be used to increase the disease resistance of many crops. In just a few years, the CRISPR technology was used to edit the wheat genome, making it resistant to bacterial blight and making corn, soybeans, and potatoes resistant to herbicides. In 2014, Chinese scientists in the Chinese Academy of Sciences used CRISPR and TALEN to simultaneously modify six copies of the wheat Mlo gene to make it resistant to powdery mildew. More and more crops are gaining profits and CRISPR technology, making it more tenacious vitality.
At the same time, CRISPR’s role in the advancement of plant biology research is also 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 introduce mutations in genes more quickly and more efficiently, destroy the coding regions of genes, and induce them to produce dysfunctional proteins, thereby exploring the function of genes. 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 genes, or even use dCas9 to regulate plant gene transcription.
In addition to food crops, CRISPR technology can also have a profound impact on livestock husbandry, and small-scale genomic modifications will greatly increase meat production in farmed animals. And similar to the application of crops, the use of CRISPR technology can easily modify multiple animal genes at the same time. For example, Chinese scientists use CRISPR to modify the myostatin gene MSTN and the growth factor gene FGF5 that can control hair growth. Goat meat production and hair quality.
Although CRISPR-edited animals can drive the development of animal husbandry, experimental animal research can best represent the unlimited potential of CRISPR technology. Whether it is used for pathological research or new drug evaluation, experimental animals are of great significance to modern medicine. The most basic of experimental animal research, the most important is to obtain a reliable animal model, in order to simulate the external manifestations and intrinsic pathogenesis of human disease.
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 strains of mouse strains used to study diseases ranging from cancer to cardiovascular disease and blindness. The emergence of CRISPR technology has provided efficient and rapid technical means for the establishment of mouse models. Not only applies to almost all mouse strains, but also can greatly shorten the time required, while its cost is relatively low, the cost is only about one-tenth of the traditional genetic tools.
Although the mouse animal model is widely used, it still has a series of limitations. For diseases such as cystic fibrosis, Parkinson’s disease, Alzheimer’s disease, etc., they are usually unable to show the characteristic symptoms of the disease, and atypical reactions may also occur in the evaluation of drug efficacy. This has made it difficult for laboratory research to transform into clinical trials. The emergence of CRISPR technology has made the establishment of non-human primate models more efficient.
Although it was possible to transfer foreign genes into the monkey genome by virus more than ten years ago, the CRISPR technology was not able to edit monkey genomes until the advent of CRISPR technology. In early 2014, Huang Xingxu of the Institute for Animal Studies at Nanjing University injected CRISPR into a single-cell embryo. The mutation in the combination of Ppar-gamma and Rag1 was used to precisely modify the genome of cynomolgus monkeys. In addition to monkeys.
CRISPR-Cas9 Horse Baby. by MichaelCammer
In addition to mice and monkeys, pigs have also become an important animal model due to the emergence of CRISPR technology. The pig’s anatomy is similar to humans in that its organ size is similar to that of humans. It also has a short reproductive cycle and a large number of litters. Therefore, 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 long been the dream of some scientists, but it is out of reach due to technical limitations. Even organ transplants between humans may produce severe immune rejection, not to mention transplants between heterogeneous species. Moreover, the existence of pig endogenous retroviruses (PERVs) is also a great security risk.
The emergence of CRISPR technology has taken us a giant step toward the pursuit of pig organs as a source of human organ transplantation. Previous techniques have largely escaped immune rejection by transferring certain genes to the pig genome, but gene editing techniques, including CRISPR, can directly knock out genes that cause immune reactions or knock out PERVs directly.
In 2015, eGenesis, founded by Harvard University’s George Church and his student Yang Lan, hopes to use CRISPR technology to realize the grand goal of pig human organ transplantation. In the same year, they achieved the feat of using CRISPR to knock out the 62 loci of PERVs in the pig genome. Then they completed another achievement: Obtaining piglets without the PERVs gene. They first knocked out the PERVs gene of the porcine embryonic connective tissue cell genome without triggering apoptosis, and then used cloning techniques to transfer the nucleus into pig ovary cells. After the embryos were developed, they were transplanted into pig uteri and successfully delivered pigs. kid.
Although the application of porcine organs still has a long way to go, the infinite potential of CRISPR allows us to change the sight of seeing this earlier. Due to her contribution to the medical field, she was selected as the Young Global Leader 2017 by the World Economic Forum.
In addition to the related applications in the medical field, the brain-drenched George Church is also working on a very well-known project: the resurrection of mammoths. The two mammoth specimens that died on both occasions between 20,000 and 60,000 years ago provided the possibility of sequencing their entire genome. Genome analysis can be used to obtain genomic changes in mammoths and existing elephants, and found that 1668 genes can be edited for proteins associated with body temperature perception, skin and hair development, and adipose tissue production. The George team used CRISPR technology to successfully replace 14 of the modern elephants with mammoth genes in 2015. However, replacing all the genes will undoubtedly make for 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 explaining in detail this magnificent goal. This book has a great deal of insight, including how to synthesize human “chiral isomers” and other research projects).
Resurrection of Mammoth By NBC
In addition to these applications, scientists are also using CRISPR technology to control genetic processes and modify the genetic information of offspring. This technology is called Genedrive. In the sexual reproduction of diploid organisms, offspring obtain a set of chromosome copies from both parents, which means that the parental gene (except for the selfish gene) is 50% likely to be passed on to offspring. But using Genedrive technology can change the way genetic information is transmitted.
Leading the project was Kevin Esvelt from George Church. The core of this technology is the knock-in of genes, using CRISPR technology to precisely cut specific sites and insert new sequences. The inserted sequence contains information for generating the CRISPR gene editing system, so it can automatically copy itself to another chromosome so that all the offspring’s chromosomes contain information that can encode the CRISPR system.
If the inserted sequence contains not only CRISPR information but also other information, then the information can also be rapidly diffused in offspring. For example, using Genedrive to insert a Plasmodium resistance gene into a mosquito genome, theoretically all mosquitoes in this area will carry all the Plasmodium resistance genes in a certain period of time. This will be a major advance in the prevention of malaria disease. However, many scientists did not stop there. They envisioned inserting genes related to female sterility in the mosquito genome, allowing the gene to rapidly spread as a virus in certain mosquito populations, resulting in the rapid extinction of the mosquito population in this area.
This can be a very terrible technology. Scientists should also do their best to avoid the spread of genetically modified mosquitoes to the outside world during laboratory experiments. It is difficult to predict the extent to which the influence will spread in the process of release, and if the mosquitoes in some areas are quickly extinct, although some scientists claim that they will not have serious impact, individuals think that the ecological impact will be difficult to predict. Ecosystems are not as simple as some models predict.
In addition, the most important issue is how to prevent the malicious use of the technology? Genedrive’s design is not very difficult. If someone inserts certain malignant genes into the mosquito’s genome, the technology will immediately become a Gene Bomb. How to safely use this technology will be a very big problem.
Preclinical experimental studies of various animal models have demonstrated the great potential of CRISPR in preclinical animal models and also provide an important tool for disease research and drug development. But can CRISPR technology be more directly used to treat diseases?
In 2013, less than a year after Zhang Feng and George Church’s laboratory proved the feasibility of CRISPR editing of human cells, the Li Jinsong team of the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences used the same gene editing tools to verify its treatment of genetic diseases. potential. They selected a mouse cataract model for genetic studies. The experimental mice in the model carried a dominant mutation in the Crygc gene that produced denatured lens proteins that caused turbidity that led to cataracts. Results CRISPR can find and repair mutations in about 2.8 billion base pairs from the mouse genome.
The researchers designed a guide RNA targeting the mutant Crygc gene and directly injected the targeting RNA and Cas9 into heterozygous fertilized eggs, and found that one third of newborn mice were cured of cataracts. And the cured mice can pass the Crygc gene repaired by the CRISPR-Cas9 system to the next generation through the germ cells. In the following years, scientists also used CRISPR to cure mice with muscle atrophy and hepatic metabolism-related diseases, and demonstrated the potential of the technology in treating major diseases such as sickle cell anemia, hemophilia, cystic fibrosis, and severe combined immunodeficiency. . Whether it is a nucleotide mutation, 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 for the direct application of human genetic diseases. Of course, the potential of CRISPR is far more than just for the treatment of genetic diseases. Scientists are still trying to use genetic editing to prevent human cells from being infected with viruses. In fact, the first genetic editing clinical trial was to treat HIV infection. In addition to infectious diseases, cancer is also one of the areas of application of CRISPR technology.
Genetic Edit By Gloria Pizzilli
Although genetic editing is a very powerful tool, it is not easy to transform 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 or embryonic?
By modifying single-cell embryos, the genome of all cells after their development will be changed, and the genomes inherited by their offspring will also be changed. Experiments on animal models have also proved the feasibility of this strategy. But this method faces this serious ethical problem. So the somatic cell became the first choice for most scientists. The genome modification of somatic cells cannot be passed on to the offspring and thus reduces ethical issues. However, the genome editing of somatic cells is far more complex than the theory of germ cells. Therefore, we must solve the new problems brought about by the selection of somatic cells for genome editing.
The biggest problem is the delivery of drugs. 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. Selecting a relatively high internal circulation system is relatively easy. The circulatory system also has two types of treatment: in vivo and in vitro gene editing (ex vivo). Relatively speaking, in vitro gene editing methods are more convenient and beneficial to quality control. In 2016, the Lu-U team of West China Hospital of Sichuan University initiated the world’s first CRISPR human clinical trial and knocked out the star molecule PD-1 on the surface of T-cells. What the team uses is the way in vitro gene editing.
But not all scientists think the same way as above. First, they use gene editing of somatic cells. In March 2015, five scholars published an article in Nature’s joint publication Don’t edit the human germ line (Don’t edit human germ cells, it sounds like a three-body listener responds to Ye Wenjie’s three sentences not to answer), and calls for scientific researchers to be cautious. Use gene editing tools to edit the germ cell genome. But only a month later, Huang Jun of Sun Yat-sen University went online and reported on the use of CRISPR technology to edit 86 inactive human embryos in order to modify HBB genes that can cause thalassaemia. Although the results of the experiment are not ideal, the article has caused great controversy internationally due to ethical issues. Many people worry that if CRISPR is used to modify human embryonic genomes to prevent hereditary diseases, the technology will inevitably be applied to modify non-medical related genetic problems. Despite this, Huang Jun was selected by Nature Magazine as the top ten scientific figures of the year.
Editing human embryos: Who is playing God’s role? (Genesis fresco by Michelangelo)
Due to the great controversy caused by the article, in the same year, the United States, Britain, and China jointly organized the Human Genome Editor International Summit in Washington to discuss the security issues, ethical issues, and government oversight of human genome editing. After this, the ethical controversy of embryonic gene editing seems to have begun to become less fierce. In 2016, Sweden and the United Kingdom became the two countries outside China that allowed embryos to perform gene editing.
As technology becomes more and more mature, there are more and more researches and articles in this field. Up to now, there have been 8 articles edited by human embryos, of which 5 were published in the past two months. Two weeks ago (September 22nd), another article by Huang Jun on the line, also modified HBB gene for the treatment of thalassaemia, but this time is the use of cloned embryonic cells, and the use of non-cutting function The CRISPR system (and carries cytidine deaminase) performs base editing to modify point mutations.
There is no doubt that the use of CRISPR for human embryonic genome editing can have a tremendous impact on the prevention of human diseases, although the current research focuses mainly on the somatic cell genome editing to treat diseases, but as technology continues to advance, CRISPR editing reproduction The potential of cells will be further explored, and ethical issues may also be better resolved.
Streptococcus thermophilus can convert lactose into lactic acid, so it is commonly used in the dairy industry. Danisco of Denmark first proved that the function of the CRISPR sequence contained in Streptococcus thermophilus is bacterial adaptive immunity to resist phage invasion. In 2011, DuPont acquired Danisco and began researching how to use CRISPR to fight bacteriophage-infected Streptococcus so that better yogurt and cheese can be made. In the same year, when Jennifer Doudna was still studying the first CRISPR system, she participated in the creation of Caribou Biosciences and hoped to use CRISPR technology to simplify the process of virus detection.
Emmanuelle Charpentier also gradually had the idea of creating a company in 2012. Five months after the Science article went live, she discussed the commercial potential of CRISPR with Rodger Novak, an old friend of Sanofi at the time, and Shaun Foy, another venture capitalist’s old friend. A month later, Novak decided to resign and co-found a new company. After that, the three people began actively looking for partners.
After the exchange with Jennifer, they plan to jointly establish this company with George Church and Zhang Feng, with a view to simplifying possible future patent issues. Unfortunately, negotiations after various known and unknown reasons have been extremely unsuccessful. A year and a half after the publication of Zhang Feng and Church articles, CRISPR technology has become more and more mature, capital is strongly expected to intervene, and the need to establish a company is becoming more and more urgent.
CRISPR Patent Hearing;Source: Science
However, under the consideration of intellectual property rights, academic credibility, geographical factors, media coverage, Nobel Prizes, and commercial returns, the four people who have made tremendous contributions to CRISPR technology have not only failed to unite but have begun to fall apart and become independent. The relationship between Jennifer and Emma’s duo is not as simple as it once was, and it is becoming more and more subtle. Not only the interests of individuals are mixed in, but the interests of the academic institutions of the University of California, Broad Institute, Harvard University, Massachusetts Institute of Technology, and University of Vienna also make the situation more and more complex.
After that, Emmanuelle Charpentier, Rodger Novak, Shaun Foy, and Chad Cowan co-founded CRISPR Therapeutics. Zhang Feng, George Church, Jennifer Doudna co-founded Editas Medicine. Erik Sontheimer, Luciano Marraffini, Derrick Rossi, and Rodolphe Barrangou co-founded Intellia Therapeutics. Other companies that later entered the market would have to pay high fees for these patents and the Broad Institute.
One month after the key patent was granted to Zhang Feng in 2014, Jennifer left Editas and joined Intellia.
I think it is very difficult now to find a biology student who has not heard of the term CRISPR. Within a few years, CRISPR technology has provided a tremendous impetus to the development of basic research fields and to the development of areas related to public life, including health care and agriculture. In the course of scientific development, there are occasional major advances. For example, relativity, such as DNA double helix, such as PCR technology, although CRISPR may not be able to par with the above breakthroughs, its impact on scientific development is beyond doubt.
Jennifer and Emma’s work provided the basis for the birth of CRISPR as a gene editing tool, and Zhang Feng and Church also demonstrated the enormous potential of CRISPR editing of mammalian cells. Undoubtedly, these people have made important contributions to the birth and development of CRISPR technology. But if there is no Jennifer, if there is no Emma, or no Church, no Zhang Feng, CRISPR technology will not appear? Obviously not (see the appendix for the timeline).
There is little controversy over the strength of CRISPR’s ability to win the Nobel Prize, but who can finally win the Nobel Prize is a big mystery. The scientific community is not as simple as people think. In the face of interests and honors, most of them become irrational. In the war on CRISPR, someone got money, someone got glory, someone was happy, but some people were lost.
If CRISPR technology has promoted the progress of science and made us look forward to a better life for the future, then the battle of interest and the battle for glory of the CRISPR technology have made us understand the human nature.
CRISPR Me. by Michele Tragakiss
Appendix: CRISPR Timeline
CRISPR Discovery and Its Function
- In 1987, Shinya Ishihara of Osaka University in Japan discovered the CRISPR sequence for the first time.
- In 1993, Francisco Mojica of the University of Alicante of Spain first identified the site that is now called CRISPR.
- In 2000, Francisco Mojica discovered and reported some common features of the previously discovered differential repeat sequence (he used the term CRISPR in communication with Ruud Jansen, who in the 2002 article officially used the term ).
- In 2005, Francisco Mojica found that the sequence matches certain segments of the phage genome and speculated that CRISPR belongs to the adaptive immune system. Another group also independently reported a similar study in the same period.
The discovery of Cas9 and PAM
In May 2005, Alexander Bolotin of the French National Agricultural Research Institute (INRA) discovered a distinctive CRISPR site during the study of Streptococcus thermophilus. The CRISPR system lacks the previously known cas protein gene, and One of the other new cas protein genes that has not been reported is one that codes for the cas gene, later named Cas9 protein. Moreover, he found that the gap sequence that matches the virus DNA contains an identical sequence PAM (protospacer adjacent motif) on one end, and PAM is necessary for the recognition of the CRISPR target sequence.
Adaptive immunity hypothesis
In March 2006, Eugene Koonin of the National Center for Biotechnology Information of the United States calculated and analyzed orthologs, and proposed the hypothesis that the CRISPR Cascade is a bacterial immune system based on the insertion of phage homologous DNA into spacers, abandoning previous concerns about the Cas protein. Assumption of the DNA repair system.
Experimental validation of adaptive immune function
In March 2007, Philippe Horvath of Danisco Company tried to study the response of phage to Streptococcus thermophilus widely used in the dairy industry. Horvath and colleagues found that the CRISPR system does indeed belong to adaptive immunity: the new phage DNA fragment was integrated into the CRISPR and used to resist the next attack of the phage. They also found that Cas9 may be the only protein required to inactivate the invading phage.
Spacer sequence is transcribed as guide RNA
In August 2008, John van der Oost of the Wageningen University in the Netherlands began to gradually analyze the CRISPR-Cas system to interfere with the process of phage invasion. Johnvan der Oost and colleagues found that phage-derived spacers can transcribe small RNAs and become CRISPR RNAs (crRNAs) that can direct Cas proteins to target genes.
How CRISPR Works on DNA Targets
In December 2008, Luciano Marraffini and Erik Sontheimer of Northwestern University in the United States discovered that the target of systemic action is DNA rather than RNA. Because people have long believed that CRISPR and RNAi have similar mechanisms, this discovery surprised researchers. Marraffini and Sontheimer said that if the system can be transferred to a non-bacterial system, it may develop into a powerful tool (note that some CRISPR systems can target RNA.
Cas9 cuts target DNA
In December 2010, Sylvain Moineau and colleagues at Laval University in Canada discovered that they can target DNA and cause double strand breaks at precise sites (three nucleotides upstream of PAM). They also confirmed that Cas9 is the only protein required for the CRISPR-Cas9 system cleavage process, and the interference process mediated by a single protein (here Cas9 protein) and crRNAs is a unique feature of the second type CRISPR system.
Discovery of tracrRNA in Cas9 System
In March 2011, Emmanuelle Charpentier’s team completed the last piece of the puzzle to resolve the mechanism of the CRISPR-Cas9 system-mediated interference process. They found that in addition to crRNA, a second RNA, called tracrRNA (trans-activating CRISPR RNA), was discovered by sequencing of S. pyogenes containing the CRISPR-Cas9 system. This RNA forms double strands with crRNA and mediates Cas9-targeted DNA targets.
CRISPR System Can Play a Role in Xenobiotics
In July 2011, Virginijus Siksnys and colleagues at the University of Vilnius, Lithuania, cloned the entire CRISPR-Cas site of P. thermophilus (containing a second type of CRISPR system), and in E. coli (without the second CRISPR system ) was expressed and found to be qigong resistant. The above experiments show that they have independent functions, and the components needed for the second type of system have all been found.
Cas9-mediated Shearing Process
- In September 2012, Virginijus Siksnys of the University of Vilnius, Lithuania, and colleagues used the above system of expression in E. coli to confirm the mechanism of action of Cas9. They determined the role of PAM as well as the cleavage site and found that the RuvC domain was able to cleave non-complementary strands by point mutations, while the HNH domain was able to cleave complementary strands. At the same time, it was shown that the crRNA sequence only requires at least 20 nucleotides, and more importantly, they found that by changing the crRNA sequence, Cas9 could be guided to target the corresponding DNA site.
- In June 2012, Emmanuelle Charpentier and Jennifer Doudna of the University of California, Berkeley, and Virginijus reported the same findings at almost the same time (Jinek et al., 2012, which was submitted later than the Gasiunas et al. article). However, they found that crRNA and tracrRNA can fuse to form one RNA, which simplifies the system. At the same time they reported the use of the system to cleave GFP DNA.
Validation of genomic editing using CRISPR-Cas9
In January 2013, Harvard- OMA Broad Research Institute and Feng Shui Institute of Harvard University George Church reported in the same issue of the Science Journal that the use of the CRISPR-Cas9 system successfully performed genome editing in eukaryotic cells. Their research shows that the CRISPR-Cas9 system is able to cleave multiple sites in the genome of human and mouse cells and can direct homologous recombination repair.