[China Pharmaceutical Network Technology News] Whenever there is a report related to CRISPR–Cas9 in the media, Addgene employees will find that the company is a non-profit company, and the research authors usually use the molecular tools they often use. Place it here so that other scientists can quickly access the tool to facilitate research. Since 2013, Addgene's phone has been ringing. When scientists first reported using the CRISPR–Cas9 system to cut the human cell genome at a specific site, other researchers were “sitting,†Initially, molecular biologists have used this new gene editing technology to change the genome of almost any organism; Addgene has sent 60,000 CRISPR-related molecular tools to researchers in 83 countries, accounting for approximately 60,000 At 17% of the total number of tools, Addgene's related pages were also viewed more than 1 million times in 2015.
The current talk about CRISPR–Cas9 technology has evolved to use this technology for human embryo editing, but researchers say that true technological innovation is just beginning; what can CRISPR bring to us? Why are scientists so keen on the technology? Because it can target specific DNA sequences of any genome, editing DNA is only one of its many roles; Daniel Bauer, a hematologist from Boston Children's Hospital, says that for many molecular biologists In other words, this method can greatly help us understand the working mode of the genome. The CRISPR–Cas9 technology can really help us understand what we want to know.
In this article, Nature has analyzed five methods, how the CRISPR–Cas9 technology helps biologists edit and modify cells.
"broken scissors"
The CRISPR–Cas9 system has two main components: the Cas9 enzyme, which prunes along the DNA like a molecular scissors; the other is a small RNA molecule that directs the trimming of scissors into a special sequence of DNA to make the incision, the original of the cell. DNA repair systems will patch these gaps extensively, but usually produce certain errors. In 2012, researchers revealed how these genetic editing tools cut human DNA, and one of the groups decided to adopt a different approach. The first thing researchers have to do is to break the genetic scissors.
Jonathan Weissman, a researcher from the University of California, said that we learned how to break the genetic scissors from Stanley Qi, a researcher at Stanford University. The researcher Stanley Qi mutated the Cas9 enzyme so that it can be at the same position as the targeting RNA. The DNA binds without cleavage of the DNA, and then the Cas9 enzyme stops and blocks the translation of other proteins. This attack system can help the researcher close the specific gene without changing the structure of the DNA sequence.
The researchers then took away the dead Cas9 enzyme and tried something new, combining the Cas9 enzyme with another protein that activates gene expression. After some clever adjustments, the researchers built a new type. The method of switching genes. At present, many laboratories have published relevant results of the modification of this method, and many researchers are competing to use this method to accelerate research progress. One of the most popular applications is to rapidly generate hundreds of different cell lines, each A cell line contains different guide RNAs that can target specific genes.
Martin Kampmann, a researcher from the University of California, San Francisco, hopes to screen every cell to see if the opening or closing of a particular gene affects the survival of neurons exposed to protein aggregates. Protein aggregation is thought to be a lot of nerves. The root cause of degenerative diseases, such as Alzheimer's disease; at the same time, the researcher Kampmann used RNA interference to conduct a similar screening study, but the results show that the technology has certain shortcomings; RNAi technology has certain off-target effects. Weissman and colleagues then adjusted the method so that it relied on long-chain guide RNAs that carry motifs that bind different proteins, and this adjustment can help scientists in the same experiment. Three different sites in the act to activate or inhibit gene expression; the researchers believe that the system can help five experiments at a time, and the only limitation is how to direct how RNAs and proteins are filled into cells.
Epigenetics of CRISPR
When geneticist Marianne Rots started his career, she wanted to develop new medical therapies, so she learned about gene therapy, a gene that targets mutations in disease, but a few years later she decided to change her own. Professional, Rots said, I realized that many diseases are caused by changes in gene expression characteristics, but I have never paid attention to one of these gene mutations, and the best way is to control the activity of the gene, so I will study The focus shifts from the genome to the epigenome.
The epigenome is a collection of "constellations" of numerous compounds added to DNA and DNA-encapsulated proteins (histones) that control the "access" to DNA, thereby regulating the proteins required for gene expression in a timely manner, and these tables The marker can change over time, which can be added or removed as the organism develops or the environment changes. In the past many years, the scientific community has spent a lot of money to catalogue epigenetic markers of different human cells, but in addition to the ability to change markers at specific sites, scientists are not sure whether they can trigger specific Biological changes, CRISPR–Cas9 technology seems to reverse this situation. In April 2015, Duke University researcher Charles Gersbach developed a system that uses broken molecular scissors to carry enzymes to specific positions in the genome. In order to add a special epigenetic marker, acetyl, to histones.
The team found that adding acetyl groups to DNA-associated histones was enough to make the expression of target genes soar. When the results were published, the researchers Gersbach "stored" the enzymes they used in Addgene for other researchers. It can also be used for related research. Gersbach predicts that when multiple epigenetic markers are manipulated simultaneously, it may lead to a synergistic effect in several research papers.
The tools provided by Gersbach need to be “refinedâ€, and many enzymes can produce or eliminate genetic markers on DNA. Researcher Rots uses zinc finger proteins to explore new functions of epigenetic markers on cancer-related genes. Today scientists use CRISPR–Cas9 to do the work, and the use of new tools will have broader effects; people may It is contingent to say that this connection, but Rots said that if the rewriting of epigenetics may not have any effect on gene expression, but now we can easily detect it, and many people are Joined in one after another.
Cisco's password deciphering
Epigenetic markers on DNA are not just broken genetic codes. More than 98% of the human genome does not encode proteins, but researchers believe that these large amounts of seemingly junk DNA actually play important. Role, and researchers are currently using the CRISPR–Cas9 technology to study the mysteries of these junk DNA. Some of these genomes edit RNA molecules, such as microRNAs and long-chain non-coding RNAs. In addition to making proteins, these RNA molecules are thought to have other functions, while other sequences are enhancers that amplify gene expression. Many DNA sequences are directly related to the risk of developing common diseases, depending on the specific genomic region containing non-coding RNAs and enhancers. Before the emergence of CRISPR–Cas9 technology, it was difficult for researchers to determine which genome problems, but now It is very convenient.
Now researchers Farnham and colleagues use CRISPR–Cas9 technology to eliminate the enhancer regions of mutations found in prostate cancer and colon cancer. In this unpublished study, the researchers removed enhancements that were considered critical. Son; David Gifford, a researcher from MIT, and Richard Sherwood, a researcher at Brigham and Women's Hospital, collaborated to use the CRISPR–Cas9 technology to trigger mutations in a 40,000-letter sequence, and then they tested whether each mutation would be nearby. The genes that produce fluorescent proteins have an effect, and the researchers mapped the results into a DNA sequence map that shows enhanced gene expression.
In-depth study of "dark matter" presents certain challenges, and the CRISPR–Cas9 technology works the same. The Cas9 enzyme cleaves a specific site for targeting RNA, but only when a special but common DNA sequence appears near the cleavage site. This way; researchers are now collecting "relatives" of the Cas9 enzyme in the bacterial kingdom, and the Cas9 enzyme recognizes different sequences; last year, Feng Zhang, a researcher at the MIT Bode Institute, discovered an enzyme family called Cpf1. This enzyme is similar to the Cas9 enzyme, which expands the selection of this particular enzyme recognition sequence; however, the researchers point out that there are currently no new enzymes that can replace the Cas9 enzyme. In the future, they hope to be able to collect completely in the genome. An enzyme that is targeted by a site.
CRISPR saw the light
Researchers Gersbach's lab uses genetic editing tools to help understand cell fate changes, and researchers have revealed how to change the fate of cells. They hope that one day they can grow tissue in culture dishes for drug screening and cell therapy development; But the effects of CRISPR–Cas9 technology are persistent, and researchers need to briefly turn genes on or off at specific sites in the tissue.
Gersbach and colleagues used this fragmentation-modifying enzyme, Cas9 (the expression of the activating gene) and the added protein activated by blue light, to induce gene expression when exposed to light. Gene expression is blocked when there is no light; Moritoshi Sato, a researcher from the University of Tokyo, developed a similar system and also created a Cas9 enzyme that can edit the genome under blue light stimulation.
Model CRISPR
In the first year of his postdoctoral research, cancer researcher Wen Xue began researching and manufacturing transgenic mice that can produce a mutation in some human liver cancers. He worked hard and finally developed a tool that is necessary for gene targeting. The tool was injected into embryonic stem cells and managed to make mice bearing mutations. The cost of the study was one year, plus $20,000. A year later, when he started another experiment on transgenic mice, his mentor tried to use the CRISPR–Cas9 technology. This time, Xue only used tools to study single-cell mouse embryos, and only in a month. The type of mouse that I wanted was obtained, and finally Wen Xue's postdoctoral career was shortened for a long time.
Researchers now studying cancer or studying neurodegenerative diseases are beginning to use CRISPR–Cas9 to create animal models for disease research that can help scientists create multimodal animals in a variety of complex ways. Xue is one of them. He used this technique to study disease mutation models in cells and cells in animals. Researchers hope to mix and pair the new CRISPR–Cas9 technology to more accurately manipulate the genome and epigenome of animal models, which will help researchers understand the complexity and pathogenesis of common human diseases.
In the case of tumors, there are many mutations that can promote cancer, the researchers Dow said, but they are not the most important for making tumors, but it is clear that we need two or three or even four mutations. To truly mimic malignant disease and to mimic human cancer more deeply; introducing all of these mutations into mice in an old-fashioned way can be costly and time consuming.
Researcher Patrick Hsu started his own research at the Salk Institute in 2015. His goal was to use genetic editing techniques to simulate neurodegenerative diseases in cell culture fluids or marmosets, such as Alzheimer's disease and Parkinson's disease, etc., can more accurately reflect the progression of human disease than the mouse model, but it is very expensive and slow before the emergence of CRISPR–Cas9 technology.
When the researcher Patrick Hsu designed a new experimental method to genetically engineer his first CRISPR–Cas9 marmoset, he said that this method may be the cornerstone for the next step of research, many technologies are instantaneous Elapsed, and we should always need to consider the biological source problem that needs to be solved. The development of future new CRISPR gene editing technologies will shine.
The current talk about CRISPR–Cas9 technology has evolved to use this technology for human embryo editing, but researchers say that true technological innovation is just beginning; what can CRISPR bring to us? Why are scientists so keen on the technology? Because it can target specific DNA sequences of any genome, editing DNA is only one of its many roles; Daniel Bauer, a hematologist from Boston Children's Hospital, says that for many molecular biologists In other words, this method can greatly help us understand the working mode of the genome. The CRISPR–Cas9 technology can really help us understand what we want to know.
In this article, Nature has analyzed five methods, how the CRISPR–Cas9 technology helps biologists edit and modify cells.
"broken scissors"
The CRISPR–Cas9 system has two main components: the Cas9 enzyme, which prunes along the DNA like a molecular scissors; the other is a small RNA molecule that directs the trimming of scissors into a special sequence of DNA to make the incision, the original of the cell. DNA repair systems will patch these gaps extensively, but usually produce certain errors. In 2012, researchers revealed how these genetic editing tools cut human DNA, and one of the groups decided to adopt a different approach. The first thing researchers have to do is to break the genetic scissors.
Jonathan Weissman, a researcher from the University of California, said that we learned how to break the genetic scissors from Stanley Qi, a researcher at Stanford University. The researcher Stanley Qi mutated the Cas9 enzyme so that it can be at the same position as the targeting RNA. The DNA binds without cleavage of the DNA, and then the Cas9 enzyme stops and blocks the translation of other proteins. This attack system can help the researcher close the specific gene without changing the structure of the DNA sequence.
The researchers then took away the dead Cas9 enzyme and tried something new, combining the Cas9 enzyme with another protein that activates gene expression. After some clever adjustments, the researchers built a new type. The method of switching genes. At present, many laboratories have published relevant results of the modification of this method, and many researchers are competing to use this method to accelerate research progress. One of the most popular applications is to rapidly generate hundreds of different cell lines, each A cell line contains different guide RNAs that can target specific genes.
Martin Kampmann, a researcher from the University of California, San Francisco, hopes to screen every cell to see if the opening or closing of a particular gene affects the survival of neurons exposed to protein aggregates. Protein aggregation is thought to be a lot of nerves. The root cause of degenerative diseases, such as Alzheimer's disease; at the same time, the researcher Kampmann used RNA interference to conduct a similar screening study, but the results show that the technology has certain shortcomings; RNAi technology has certain off-target effects. Weissman and colleagues then adjusted the method so that it relied on long-chain guide RNAs that carry motifs that bind different proteins, and this adjustment can help scientists in the same experiment. Three different sites in the act to activate or inhibit gene expression; the researchers believe that the system can help five experiments at a time, and the only limitation is how to direct how RNAs and proteins are filled into cells.
Epigenetics of CRISPR
When geneticist Marianne Rots started his career, she wanted to develop new medical therapies, so she learned about gene therapy, a gene that targets mutations in disease, but a few years later she decided to change her own. Professional, Rots said, I realized that many diseases are caused by changes in gene expression characteristics, but I have never paid attention to one of these gene mutations, and the best way is to control the activity of the gene, so I will study The focus shifts from the genome to the epigenome.
The epigenome is a collection of "constellations" of numerous compounds added to DNA and DNA-encapsulated proteins (histones) that control the "access" to DNA, thereby regulating the proteins required for gene expression in a timely manner, and these tables The marker can change over time, which can be added or removed as the organism develops or the environment changes. In the past many years, the scientific community has spent a lot of money to catalogue epigenetic markers of different human cells, but in addition to the ability to change markers at specific sites, scientists are not sure whether they can trigger specific Biological changes, CRISPR–Cas9 technology seems to reverse this situation. In April 2015, Duke University researcher Charles Gersbach developed a system that uses broken molecular scissors to carry enzymes to specific positions in the genome. In order to add a special epigenetic marker, acetyl, to histones.
The team found that adding acetyl groups to DNA-associated histones was enough to make the expression of target genes soar. When the results were published, the researchers Gersbach "stored" the enzymes they used in Addgene for other researchers. It can also be used for related research. Gersbach predicts that when multiple epigenetic markers are manipulated simultaneously, it may lead to a synergistic effect in several research papers.
The tools provided by Gersbach need to be “refinedâ€, and many enzymes can produce or eliminate genetic markers on DNA. Researcher Rots uses zinc finger proteins to explore new functions of epigenetic markers on cancer-related genes. Today scientists use CRISPR–Cas9 to do the work, and the use of new tools will have broader effects; people may It is contingent to say that this connection, but Rots said that if the rewriting of epigenetics may not have any effect on gene expression, but now we can easily detect it, and many people are Joined in one after another.
Cisco's password deciphering
Epigenetic markers on DNA are not just broken genetic codes. More than 98% of the human genome does not encode proteins, but researchers believe that these large amounts of seemingly junk DNA actually play important. Role, and researchers are currently using the CRISPR–Cas9 technology to study the mysteries of these junk DNA. Some of these genomes edit RNA molecules, such as microRNAs and long-chain non-coding RNAs. In addition to making proteins, these RNA molecules are thought to have other functions, while other sequences are enhancers that amplify gene expression. Many DNA sequences are directly related to the risk of developing common diseases, depending on the specific genomic region containing non-coding RNAs and enhancers. Before the emergence of CRISPR–Cas9 technology, it was difficult for researchers to determine which genome problems, but now It is very convenient.
Now researchers Farnham and colleagues use CRISPR–Cas9 technology to eliminate the enhancer regions of mutations found in prostate cancer and colon cancer. In this unpublished study, the researchers removed enhancements that were considered critical. Son; David Gifford, a researcher from MIT, and Richard Sherwood, a researcher at Brigham and Women's Hospital, collaborated to use the CRISPR–Cas9 technology to trigger mutations in a 40,000-letter sequence, and then they tested whether each mutation would be nearby. The genes that produce fluorescent proteins have an effect, and the researchers mapped the results into a DNA sequence map that shows enhanced gene expression.
In-depth study of "dark matter" presents certain challenges, and the CRISPR–Cas9 technology works the same. The Cas9 enzyme cleaves a specific site for targeting RNA, but only when a special but common DNA sequence appears near the cleavage site. This way; researchers are now collecting "relatives" of the Cas9 enzyme in the bacterial kingdom, and the Cas9 enzyme recognizes different sequences; last year, Feng Zhang, a researcher at the MIT Bode Institute, discovered an enzyme family called Cpf1. This enzyme is similar to the Cas9 enzyme, which expands the selection of this particular enzyme recognition sequence; however, the researchers point out that there are currently no new enzymes that can replace the Cas9 enzyme. In the future, they hope to be able to collect completely in the genome. An enzyme that is targeted by a site.
CRISPR saw the light
Researchers Gersbach's lab uses genetic editing tools to help understand cell fate changes, and researchers have revealed how to change the fate of cells. They hope that one day they can grow tissue in culture dishes for drug screening and cell therapy development; But the effects of CRISPR–Cas9 technology are persistent, and researchers need to briefly turn genes on or off at specific sites in the tissue.
Gersbach and colleagues used this fragmentation-modifying enzyme, Cas9 (the expression of the activating gene) and the added protein activated by blue light, to induce gene expression when exposed to light. Gene expression is blocked when there is no light; Moritoshi Sato, a researcher from the University of Tokyo, developed a similar system and also created a Cas9 enzyme that can edit the genome under blue light stimulation.
Model CRISPR
In the first year of his postdoctoral research, cancer researcher Wen Xue began researching and manufacturing transgenic mice that can produce a mutation in some human liver cancers. He worked hard and finally developed a tool that is necessary for gene targeting. The tool was injected into embryonic stem cells and managed to make mice bearing mutations. The cost of the study was one year, plus $20,000. A year later, when he started another experiment on transgenic mice, his mentor tried to use the CRISPR–Cas9 technology. This time, Xue only used tools to study single-cell mouse embryos, and only in a month. The type of mouse that I wanted was obtained, and finally Wen Xue's postdoctoral career was shortened for a long time.
Researchers now studying cancer or studying neurodegenerative diseases are beginning to use CRISPR–Cas9 to create animal models for disease research that can help scientists create multimodal animals in a variety of complex ways. Xue is one of them. He used this technique to study disease mutation models in cells and cells in animals. Researchers hope to mix and pair the new CRISPR–Cas9 technology to more accurately manipulate the genome and epigenome of animal models, which will help researchers understand the complexity and pathogenesis of common human diseases.
In the case of tumors, there are many mutations that can promote cancer, the researchers Dow said, but they are not the most important for making tumors, but it is clear that we need two or three or even four mutations. To truly mimic malignant disease and to mimic human cancer more deeply; introducing all of these mutations into mice in an old-fashioned way can be costly and time consuming.
Researcher Patrick Hsu started his own research at the Salk Institute in 2015. His goal was to use genetic editing techniques to simulate neurodegenerative diseases in cell culture fluids or marmosets, such as Alzheimer's disease and Parkinson's disease, etc., can more accurately reflect the progression of human disease than the mouse model, but it is very expensive and slow before the emergence of CRISPR–Cas9 technology.
When the researcher Patrick Hsu designed a new experimental method to genetically engineer his first CRISPR–Cas9 marmoset, he said that this method may be the cornerstone for the next step of research, many technologies are instantaneous Elapsed, and we should always need to consider the biological source problem that needs to be solved. The development of future new CRISPR gene editing technologies will shine.
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Red light therapy has been studied for its potential to improve skin health, reduce inflammation, enhance muscle recovery, and promote overall well-being. It is often used in professional settings, such as spas and wellness centers, but there are also portable at-home versions available for personal use.
Red light therapy panels can be used by positioning them close to the body or by standing in front of them. The treatment duration and frequency vary depending on the desired outcome and the specific device being used. It is generally considered a safe and non-invasive therapy with minimal side effects.
It's important to note that while red light therapy has shown promising results in some studies, more research is needed to fully understand its effectiveness and potential applications. As with any medical or wellness treatment, it is advisable to consult with a healthcare professional before starting red light therapy.
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