Posted by on February 24, 2018 7:51 am
Categories: Baidu

Source: CRISPR evolutionary history—from gene editing to gene detection

gene editing Chronicles from 30 years ago, the first gene editing in yeast cells the experiment is completed.but until the past five years, the CRISPR Cas9 system appears and quickly become the most popular gene-editing tools, is “Science” magazine as the 2015 breakthrough technology.

initially, the CRISPR-Cas9 system is only specific bacteria the body to resist viral infections in the“immune system”, however, including Feng Zhang, Professor included, many researchers are aware, this system is expected to be used for precise and rapid gene editing. In 2012, Jennifer Doudna, Professor and Emmanuelle Charpentier, Professor at the science magazine published an article, confirming the CRISPR-Cas9 system for in vitro experiments can be“fixed”to the DNA to be cut. Two months later, Virginijus Siksnys Professor in the PNAS Journal published a similar study. These papers also show that CRISPR-Cas9 system as a gene editing tool of enormous potential.

many in the industry believe that, Doudna, Professor Charpentier, Professor Zhang Feng, a Professor is expected to result in the CRISPR-Cas9 system on the outstanding contributions and received the Nobel Prizeimage source: PLOS)

this from the CRISPR-Cas9 system the further application of only the difference between the finishing touches: scientists need to confirm that this item comes from a bacterial gene editing system, the same can be applied to the human body. That’s a bold assumption. You know, both the genome complexity with a cloud of mud.

however, Zhang Feng, the Professor and his team did. In 2013, Feng Zhang, Professor in the science magazine published a blockbuster study: theyfor the first time in mammals.the application of the CRISPR-Cas9 system, and confirm it in a few weeks to establish small rodent disease model. In addition, Zhang Feng, the team alsofor the first time in human cellssuccessfully used the CRISPR-Cas9 system complete gene editing.

these findings brought the CRISPR-Cas9 system is a huge change. It let the scientists were able to complete a previously not dare to conceive the work. Today, we expect to be able to remove each of the infected cells in the HIV, or the treatment of sickle-shaped red blood cell anemia and other classic genetic diseases. Even, the scientists have the imagination to use it to overcome cancer. In addition, it can also be in the plant genome. This can bring a new bio-energy source, or bring in traits the more stable crops.

CRISPR gene editing technology Open Big Five“martial”


breaking shear

CRISPR-Cas9 technology has two main components: one is the Cas9 enzyme, as a molecular scissors-like shearing of the DNA; the other is a small RNA molecule, the“scissors”directed to a specific DNA sequence and cut. The cell’s inherent DNA repair machines will usually fix the cut, but often an error occurs.

nevertheless, it has given hope to disrupt the gene in order to understand the content of their work of the scientists has brought benefits. The gene encoding is relentless: fix the middle a little mistake will completely change its encoding of the protein sequence, or to completely stop protein production. Therefore, scientists can be in protein or gene disruption, the study of cell or organism what is happening.

but there is a different repair channel, and sometimes also according to the DNA template to repair the cut. If researchers with a template, then they will be able to choose almost any site to edit almost any desired gene sequence.

University of California, San Francisco system biologist Jonathan Weissman team hopes to learn about this gene-editing tool in the cut the human DNA aspect exactly how, but they are going to use a different method.“ We do the firstOne thing is: breaking the cut.” Weissman said. The team with the“dead”Cas9 try some new methods. Researchers to be based in another part of the protein, the protein is able to activate gene expression. Plus a few other twisted, they eventually let the gene can be according to the will of the open and close.

since then, a number of laboratory based on the method published by a different research results. The method also attracted the Massachusetts Institute of technology synthetic biologist Ron Weiss added to the CRISPR research boom. The team in a single experiment to establish a plurality of gene twist, so that it can be faster, easier to construct complex biological circuits.“ Synthetic biology the most important goal is to be able to by these exact circuit to build complex behavior.” Weiss said.



when the geneticist Marianne Rots the start of their career, she hopes to discover new medical method, through gene therapy targeting those diseases mutated gene. A few years later, in Groningen, Netherlands University Medical Center work, she decided to change the action direction. She believes that the control of gene activity is the best way to adjust the epigenome rather than the genome itself.

the epigenome is the Additional in the DNA and DNA wrapped in a protein compound that is a histone on aggregate. They can control the DNA path, open or closed leading to gene expression of the protein. While these tag will change over time as the organism development and changes in the environment, they will be added or removed.

over the past few years, millions of dollars have been invested into this field, such as calculating the different in human cells epigenetic Mark as well as with the brain activity and tumor growth associated with the genetic pattern. But since there is no ability to change a specific bit marker, the researchers could not determine whether they will result in biological changes.

CRISPR-Cas9 will reverse the situation. 2015 4 on the North Carolina bio-engineer Charles Gersbach and colleagues published a technology, the use of shear acetyl group, an epigenetic tags added to histone.

Rots had the use of a relatively old gene editing tools zinc finger of the enzyme protein to explore the epigenetic markers of the function, and now she is in the use of CRISPR-Cas9 on.“ The new tools allow this area to become democratization, and has generated a broad implications of.” She said. Rots shows, people used to say, if you re-write epigenetic genes will not have gene expression caused by the impact, or the relationship between the two is no coincidence.“ But now the test is very simple, many people are joining this field.” She said.


code decryption

DNA epigenetic marks are not only waiting for the unraveling of the genetic code. More than 98% of the human genome are not indicated proteins genetic code. Researchers believe that a large number of DNA play an important role, so they are using CRISPR-Cas9 to understand code is what.

RNA molecule of some encoding, such as microRNAs and small molecules RNA and long non-coding RNA, be considered with the production of protein does not matter, the other sequence is a sequence in which the next instruction to expand the gene expression of“Enhancer”in. Most common risk of disease-related DNA sequence located in the contain the non-coding RNAS and Enhancer genomic region. But in CRISPR technology, researchers find it difficult to understand those sequences in what to do.“ We have no good method in the functional interpretation of non-coding genome.” Bauer said,“Now, our experiments and delicate.”

as researchers use CRISPR-Cas9 technology to explore more and more conventional DNA may also occur more surprises. However, even the use of CRISPR-Cas9, to explore the unknown areas also present challenges. The Cas9 enzyme to the RNA of the wizard, under the guidance of the shear need to edit the place, but this is only when a specific, common DNA sequence is located in the cut near the point. This will give those who wish to make a gene silencing of the scientists bring a piece of the puzzle, because the key sequence is almost never present in the gene interior. Researchers are exploring the Kingdom of bacteria, looking for to be able to identify the different sequence of the Cas9 enzyme“kindred”。

last year, mit and Harvard-affiliated broad Institute, Feng Zhang lab found that one called Cpf1 enzyme family capable of spreading sequence selection. But Agami emphasized that, so far identified as Cas9 as the widespread use of enzymes is still very small. The future, he hopes to be able to Own the entire series, can be used to target the genome in any sites of the enzyme.“ We now also do not get there.” He said.


contact light

Gersbach lab is using a gene editing technology as the understanding of cell fate and how to manipulate the cells of some of the tools: the team hope that the future one day in a Petri dish to cultivate the tissue for drug testing and cell therapy. But the CRISPR-Cas9 role is permanent, Gersbach team need to from time to time open or turn off the gene, and in the organization of very specific sites.“ Analog of blood vessels requires a high degree of control.” He said.

Gersbach and colleagues to choose the irregular of the editing“scissors” – now able to activate the genes Cas9, and joined by blue light activation of the protein. When the cells exposure to light, the system is able to stimulate gene expression; but there is no light, the system will stop gene expression. By Japan’s University of Tokyo biochemist Moritoshi Sato led the team developed a similar system, and may also be in contact to Blu-ray after the activation of Cas9, to achieve gene editing.

by the CRISPR techniques with the chemical“switch”combined with other people also got similar results. New York Weill Cornell Medical College cancer geneticist Lukas Dow want in adult rats in vivo generated cancer-mutation-associated genes, which replicate in human colorectal cancer patients found a genetic mutation. The team used CRISPR-Cas9 technology, by a dose of deoxy-oxytetracycline activation of Cas9, so that it cut off the targeted goals.


disease model

from cancer to neurodegenerative diseases and other fields of researchers are working through the CRISPR-Cas9 technology to create disease in animal models. This allows researchers to more animals, in a more complex manner, in a greater range of gene editing. University of Massachusetts Medical School Cancer Research specialist Wen Xue are being systematically selected tumor gene data, the use of CRISPR-Cas9 simulation of Petri dish and animal cells in vivo growth process of mutation.

the researchers hope that by mixing matching new CRISPR-Cas9 tools, the precise manipulation of animal models of the genome and the epigenome in.“ The real power is the integration of those systems.” Dow says. This might allow scientists informed of and understand the common diseases of complex traits.

bio-engineer Patrick Hsu in 2015, the establishment of which is located in California Sacramento, Institute of Biology of the laboratory, aims to use gene editing technology in a Petri dish and marmoset body simulation of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. As will be able to than the rat model more effectively reproduce the human disease, but in the absence of CRISPR-Cas9 before, to achieve all this the cost is extremely high, and the process is extremely slow.

even in his design of experiments for the first CRISPR-Cas9 marmoset gene editing, Hsu is also very clear, this method is just a technique of“stepping stone”.“ Science and Technology of the old went and the new came again. You can’t forever and a technique to‘fall in love’with.” He said,“You should always be thinking, what kind of biological problems remain to be solved.”

human gene therapy applications

gene editing“to the species DNA into what we want”,rely on it,humans can be implemented not only animals, plants, the rapid directional breeding,but also to promote the treatment of diseases of the Subversion of the sexual revolution,access to“rewrite the life script of the magic brush”is. The not too distant future,mankind is expected to will not be able to cure the disease becomes the long-term control of chronic conditions

the United States is still studying, how will CRIPSR gene editing technologies applied to human���it. At the same time, China is the field of rapid action, trying to achieve a breakthrough.

according to the Wall Street Journal reported, from 2015 years, China has used the CRISPR-Cas9 technology, in 86 personal on the gene editing experiment. Unlike the US, China is no relevant rules and regulations, to avoid a science experiment towards the wrong direction. Therefore, the Hangzhou tumor hospital Dean Wu 琇 have been trying to use this technology to treat cancer patients.

in fact, the management of the hospital only in the afternoon, spend half an hour on the ratification of the Wu formula 琇 of the plan.

it was unclear whether these attempts whether the obtained effects. However initial reports suggest that some the test was a success. However, this is not China for the first time in humans using CRIPSR technology, and such attempts tend to produce catastrophic consequences. 2016, Chinese scientists have tried to use CRIPSR technology on Human embryonic genetic modification, but found that at least 2/3 of the occurrence of the mutations in 28 surviving embryos, only a small part(a total of 86, contains a alternative of the gene information.

according to the U.S. National Medical database of information, China has conducted at least 9 items based on the body of the CRISPR test. The Wall Street Journal also found that, from 2015 onwards, China for this technology were also at least two human trials.

▲Dr. Huang is a Professor in doing the experiment.

2016 8 months,Sichuan University, West China hospital, Cancer Center, breast cancer Department Director Lu uranium team will carry out the world’s first CRISPR-Cas9 gene editing treatment of lung cancer clinical trials. British “nature” magazine for its evaluation is“ahead of the rest of the world is being carried out gene editing test.”

although some people worry that China will take in the CRISPR technology has made the advantages, but also there have been calls for this new technology to maintain caution and patience. CRISPR technology originally developed by Jennifer Dudley’s Jennifer Doudna and Emma Newman, Charles Pang tyre(Emmanuelle Charpentier Co-found. The United States and Europe are of this conservative attitude, has yet to start human trials.

Penn West Vania University of CRISPR research team chief scientist Carl Chu’s Carl June, Dr. said:“it is difficult to judge, how to act quickly and ensure patient safety is maintained between the balance.” In addressing the many regulatory obstacles, the team will start human trials.

application to gene detection

2 May 15, “Science” published the two studies, respectively, demonstrated by Jennifer Doudna and Feng Zhang lab is based on the CRISPR system and development of new diagnostic tools.

in one of the papers, Doudna’s team demonstrated a named DETECTR system,it can accurately identify the source of the sample in different types of HPV another paper, Zhang Feng, the team is showing the performance optimization of the SHERLOCK system, which last year successfully developed,for the detection of human-derived samples in the zika virus infection, dengue virus and other harmful bacteria.

these two papers together demonstrated CRISPR great potential, not only for genome editing.

“it makes a new generation of diagnostic techniques become possible, and the ratio of the current technology is more cost-effective, theRochester College of Biochemistry and biological Physics Department assistant Professor Mitchell O’Connell said.

Doudna missionteam 100%accurately detect the HPV-16 infection

in “Science” published studies, the Doudna team used��� CRISPR-Cas12a system. They found a very interesting phenomenon: this CRISPR system in the shear-targeting of double-stranded DNA at the same time, Cas12 DNA enzyme activity can be activated, and the enzyme can be non-specific cutting of single-stranded DNA(ssDNA)。

the study’s co-author, University of California, Berkeley lab researcher Janice Chen this says:“this is an unexpected but very‘crazy’the discovery of。”

/ DETECTR works

the reason that this is a“crazy”foundbecause of this finding for intracellular detection of whether it contains a desired DNA offers a completely new idea:while the intracellular delivery of targeting the DNA of the CRISPR-Cas12a system and nonspecific ssDNA fluorescent reporter gene(FQ-labeled reporter, and upon detection of the target DNA, the CRISPR-Cas12a the system will start at the same time, a fluorescent reporter gene will also be degraded, thereby releasing the fluorescent signal.

based on this, Chen and her colleagues developed a can be used to diagnose viral infection of the new diagnostic tools, and through theisothermal nucleic acid amplificationtechnology with improved sensitivity. The tool is named DNA Endonuclease Targeted CRISPR Trans Reporter, referred to as DETECTR

so far, the scientists have been in the test tube the level of proof DETECTR can100%accurately detected HPV16 infections, 92% of accurately detected HPV18 infectionit. HPV16 and HPV18 can increase the occur in patients with cancer probability, are two particularly dangerous HPV subtypes.

“of course, we also need to constantly improve the system. But in principle, this idea is feasible, we’re really excited.” Chen said.

it is gratifying, compared to the other tests, DETECTR test is inexpensive and quick:a single cost less than a dollar, just one hours

now,Chen and her team are trying to develop can easily read the fluorescence signal of the hardware devicesit. They also want to test whether the system can respond to other human samples, such as blood, saliva or urine.

Feng Zhang team: sensitivity increase 100 times, can detect a variety of viruses

last year, Zhang Feng, the team demonstrated to the public SHERLOCK how to use the CRISPR-Cas13a in a variety of human samples, such as saliva found walled card, dengue RNA sequences, and even some with cancer mutations in the relevant sequence.

today, SHERLOCKv2 was born.with the SHERLOCK compared, the sensitivity increased 100-fold, and in the same sample detected a variety of virus infection, such asat the same time the detection of the stronghold card and the dengue virus

has been able to achieve such results is because they in SHERLOCKv2 the introduction of a variety from different species of bacteria of the genus Cas13 enzymes, such as LwaCas13a and PsmCas13b it.

/ CRISPR-Cas13a

these different Cas of the enzyme for different RNA sequences exhibit a different“preference” other words, while the cellular delivery of a variety of enzymes and a variety of different fluorescent reporter gene, once the CRISPR system found the target gene, the corresponding Cas13 it will start clipping enzyme activity, shear corresponding to the fluorescent reporter genes, thereby releasing the fluorescent signal.

this principle actually and Doudna team DETECTR basically the same, but Zhang Feng, the team designed a fluorescent reporter gene must be specific, and Doudna team is very specific.< span>however, it is also the“specificity”of this advantage so that SHERLOCKv2 can be the simultaneous detection of multiple sequences.

in the same phase��The idea is, in order to improve the sensitivity, Zhang Feng, the team also introduced the isothermal core amplification technology. But in addition, they to the system also introduces a CRISPR type-III in the Csm6 enzyme. Csm6 enzyme specific cleavage sequence for the cyclic adenosine monophosphate molecule and the end of the 2’,3’-cyclic phosphate of the linear amp. They found that by designing a CRISPR system so that Cas13 after the cut sequence is Csm6 enzyme targeting sequence, can greatly improve the sensitivity.

“these into a tube in all enzymes, they are not only interactions play a better role, but let us give the precious biological information. It’s really great full to us to show the Biochemistry of the powerful forces of”, the paper’s co-author, from Feng Zhang Lab’s PhD students, Jonathan Gootenberg said.

currently, virus infection diagnosis generally include virus isolation and identification, virus nucleic acid and antigen direct detection and specific antibody detection,is not only time-consuming, but also for medical equipment and operating personnel’s professional standards requirements are very high.therefore, if we can developed a dependence on expensive equipment and the health care provider of smart diagnostic equipment is very important, especially for viral infections often wanton rampant in the developing countries.

with the DETECTR compared to Zhang Feng team paper described a diagnostic tool SHERLOCKv2 away from this goal even further.

figure | SHERLOCK test paper test results show

like DETECTR, like, SHERLOCK also using a fluorescence signal as output.< strong>but is not limited to this, Zhang Feng, the team has also developed a similar to a pregnancy test, like the test paper, do not need any special equipment, thejust a piece of paper, the SHERLOCKv2 will be able to show the virus detection results, very easy to use.

“imagine a very bad situation, no energy such as electricity. However, as long as there are samples, SHERLOCKv2 test paper will be in the field play a role”, O’Connell said. And it’s also very cheap:SHERLOCKv2 test paper just a few dollars

although DETECTR and SHERLOCK has to we show their use in the diagnosis of a powerful force, but on entering the before clinical use, the researchers still have a lot of work needs to be done to ensure the accuracy of diagnosis.

however, I believe that these new diagnostic tools will absolutely rewrite the future of diagnostic technology, particularly to those who lack the advanced equipment and the trained personnel of developing countries is very different.“ It affects human health and society as a whole the true potential of”from Feng Zhang lab of another doctoral student, the thesis of co-author Omar Abudayyeh said.

all in all, the two teams are being developed based on these CRISPR, for the rapid diagnosis of infections cheap device,promises to be the world, especially in developing countries the virus, such as HPV and walled card infection diagnosis, brought a real revolution

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statement: this article by the settled Sohu number of authors authored, in addition to Sohu official account, views are those of the authors themselves, does not mean Sohu positions.

Published at Thu, 24 Feb 2018 07:03:29 +0000

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