2018 2 on CRISPR/Cas the latest research progress

Source: 2018 2 on CRISPR/Cas the latest research progress

2018 年 2 月 28 day/bio Valley BIOON/—genome editing technology CRISPR/Cas9 is the journal Science as 2013 annual top ten scientific and technological progress one of the subject to People’s attention. CRISPR is a regular intervals of clustered short palindromic repeats, Cas is CRISPR-associated protein referred to. CRISPR/Cas was originally developed in bacteria found in the body, is bacteria used to identify and destroy anti-phage and other pathogens invading the defensive system.

about the past 2 months, what are the major CRISPR/Cas research or discovery? Small comb a bit this month bio Valley reported the CRISPR/Cas research news, for everyone to read.

1. Two Cell indicates that the use of a method based on CRISPR/Cas9 technology is expected to the treatment of fragile X chromosome syndrome

doi:10.1016/j. cell. 2018. 01. 012; doi:10.1016/j. cell. 2016. 08. 056

fragile X chromosome syndrome is caused by the X chromosome FMR1 gene mutation caused. Methylation will prevent this occurring mutation of the gene expression. Has been confirmed in brain development, the lack of FMR1-encoded protein will cause of this syndrome associated with neuronal hyperexcitability. Now, in a new study from the Whitehead biomedical Institute researchers for the first time to use them to develop a Remove methylation improved the CRISPR / Cas9 system to restore the affected by this disease the neurons of the FMR1 gene activity, suggesting that this method might proved to be a targeted by abnormal methylation of diseases caused by useful examples. Relevant research results in 2018 2 May 15, online published in Cell Journal, paper titled“Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene”。 Thesis communication the author of the Whitehead biomedical Institute founding member Rudolf Jaenisch. p>

this study is the first direct evidence that the FMR1 gene-specific fragments to methylation can re-activate this gene, thereby saving the affected by the fragile X chromosome syndrome affects the neurons of the fragile X chromosome syndrome neurons in.

2. Science: the use of CRISPR to build a cellular event Recorder—CAMERA

doi:10.1126/science. aap8992; doi:10.1126/science. 359. 6377. 728

in a new study, from the United States, Broadbent Institute, Weixin Tang, and David R. Liu developed a way to use CRISPR to build cell event recording system technology. In their 2018 2 May 15, published online in Science Journal entitled“Rewritable multi-event analog recording in bacterial and mammalian cells,”the paper, they describe this technique and use it to develop two kinds of recording systems. Jon Cohen for this study in 2018 2 May 16, the Science journals on the discussion.

The two researchers reported that they developed a method called CAMERA（CRISPR-mediated analog multi-event recording apparatus, CRISPR-mediated simulation of multi-event recording apparatus of the technology, they are using this technology to build two types of cell recording system.

in the first one is called“CAMERA 1”in the cell of the recording system, the two researchers will be two between each slightly different from the plasmid injected into the bacterial cells, followed by exposure to the required stimulus period, the use of CRISPR-Cas9 for these two plasmids, one for cutting, thereby inducing the cells to produce another plasmid to replace it. In so doing when an event occurs recording them. This technique allows the researchers recorded how cells of the nutrients or antibiotics such as stimulus response.

in the second is referred to as“CAMERA 2”in the cell of the recording system, when the desired signal occurs in the cell, the two researchers using the base editor base editor to change the genetic code of the individual bases. Using this technique, they recorded when exposure to the virus, nutrients and antibiotics and other stimuli, the cells how to respond. They reported this technique is successfully used for bacterial cells and human cells.

3. Science: a major breakthrough! Developed based on CRISPR disposable multi -��Nucleic acid detection platform

doi:10.1126/science. aaq0179

in a new study, before the first time developed a rapid and inexpensive highly sensitive based on the CRISPR diagnostic tool—is called SHERLOCK（Specific High Sensitivity Enzymatic Reporter UnLOCKing the—the researchers greatly enhances this tool’s capabilities, and developed a micro-strip test method, this test method allows the naked eye to the test results, without the use of expensive equipment. Relevant research results in 2018 2 May 15, published online in the Science journal, The paper entitled“Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 is”.

SHERLOCK the key to success is that one is called Cas13 CRISPR-associated protein, this protein is programmed after binding to a specific RNA fragment. Cas13 of the target can be any gene sequence, including the viral genome, in bacteria that confer antibiotic resistance gene or a cancer-causing mutation. In some cases, once Cas13 positioning and cutting it to the specified target, this enzyme will into Overdrive, so the indiscriminate cutting in the vicinity of the other RNA. In order to develop SHERLOCK, these researchers will this“off-target”activity converted to its advantage, to design a such and DNA and RNA is compatible with the system.

SHERLOCK the diagnostic potential depends on the additional suffering after cutting to generate a signal of the synthesized RNA chain. In Cas13 binding to its initial target, it will cut this synthetic RNA, the release of signal molecules, thereby generating a indicate that it targets the presence or absence of the measurement value.

SHERLOCK platform are now able to apply to test a variety of targets. SHERLOCK was initially at once can only be detected by a nucleic acid sequence, but now a single analysis can simultaneously serve up to 4 different targets to provide a fluorescent signal—this means that you need less samples for the diagnosis. For example, this new SHERLOCK version can be in a single reaction in determining a whether a sample contains a zika virus infection or dengue virus particles, the virus particles will cause the patient to appear similar symptoms. This platform utilizes from different bacterial species of the Cas13 and Cas12a, formerly known as the Cpf1）enzyme to produce these extra signals.

4. Science: major progress! Developed based on the CRISPR-Cas12a techniques to detect viral DNA

a powerful genome editing tool can be used as an ACE DNA detectives were deployed to sniff out the indication of viral infections, cancer or even of the defective gene there is a DNA fragment.

Doudna said, This is called DETECTR DNA Endonuclease Targeted CRISPR Trans Reporter of DNA detective tool“to achieve sensitive and accurate DNA testing”, and can be in the human sample found that the two oncogenic human papilloma virus（HPV）。 2017 11 months, she and her colleagues first in bioRxiv. org published on the study of the Preprint doi:10.1101/226993; the relevant studies to 2018 2 May 15, published online in the Science journal, The paper entitled,“CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity is.”

pictures from the HHMI to.

the researchers observed in some cases, Cas12a will turn into a DNA shredder, any nearby single-stranded DNA to be cut. But this is not indiscriminate it. In order to start machete action, Cas12a must first find a precise DNA targets. People can by adding one to tell Cas12a looking for what the guide RNA molecule to the target can be programmed. Harrington said,“by re-programming to find that you want to detect any DNA fragment is very simple.”

once Cas12a lock it in target and cut, it will begin to tear it can find all of the single-stranded DNA. But in order for this system to become useful, Doudna and her colleagues need a method to observe Cas12a when to start this molecule killed, thereby indicating that it has found its target. Therefore, these researchers used a light-emitting molecule, an easy-to-find fluorescent molecules, and this light-emitting molecule by a single-stranded DNA with a blocking of such light-emitting molecules of the light emitting inhibitory molecules even���Together. When Cas12a start machete action, it is cutting will be of such light-emitting molecules and such inhibitory molecules are connected together to the single-stranded DNA. This would remove this inhibitory molecules, so that the light emitting elements of the light emitting—a researchers to be able to detect the signal.

The Doudna team then used their DNA detective testing. With UCSF, Joel Palefsky and Dr. and team cooperation, they’re looking for from the two oncogenic HPV—HPV16 and HPV18—DNA signal. These researchers obtained 25 parts from a not infected with HPV, infection with both oncogenic HPV in one and simultaneously infected with both oncogenic HPV human DNA samples. For HPV16, DETECTR all of 25 parts of the sample make the correct judgment. For HPV18, DETECTR correctly determined that 25 parts of a sample of 23 parts. Doudna said, it missed the sample signal is relatively weak, may be able to through the different design of the guide RNA to be improved.

5. Science: challenge routine! Based on the use of CRISPR/Cas9 to the DNA marker technology to observe the dynamics of DNA dance

doi:10.1126/science. aao3136

DNA in transcription occurs during a twitch, so far away from the genomic region of the contact, thereby enhancing gene expression. In a new study from Stanford University researchers have developed a new method to mark a single non-repetitive DNA sequence. Relevant research results in 2018 1 December 25, published online in the Science journal, The paper entitled“Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements”. p>

this new DNA marker techniques to the use of fluorescent molecules accurately labeled to any single DNA fragment, and track their three-dimensional position and motion, allowing to reveal this DNA dance. These researchers, this technology will be referred to as chimeric gRNA oligonucleotide array chimeric array of the gRNA oligo, CARGO Airport. It is the CRISPR/Cas9 gene editing tool is a variant, is expected to trigger genome to study kinetics of the transformation.

Guys, as a thesis of the first author graduate student Bo Gu and another paper co-author Tomasz Swigut, Dr devised a method: by many different gRNA composed of an array into cells, thereby accurately identifying a unique non-repetitive DNA fragments and using a variety of fluorescent molecules to mark them, so that you can under a microscope it is easy to visual observation to them.

6. Cell Rep: explain why CRISPR-Cas9 presence of off-target effects.

doi:10.1016/j. celrep. 2018. 01. 045

Cas9 protein found simplifies genome editing, and may even in the near future to eliminate many hereditary diseases. The use of Cas9, scientists were able to cut the cell DNA, thereby correcting the mutated gene, or the new genetic material introduced into this new cut of the bit point. Initially, the CRISPR-Cas9 system seems to be very accurate. However, today, it is obvious that Cas9 also sometimes cut with its targeted sequence is similar to other DNA sequences. In a new study, from the Netherlands Delft University of technology researchers have developed a mathematical model to explain why the Cas9 will cleave some DNA sequences, while allowing the other of the DNA sequence remains intact. Related research results published in 2018 2 May 6, the Cell Reports journal, The paper entitled“Hybridization Kinetics Explains the CRISPR-Cas Off-Targeting Rules”as well.

Depken explains,“when the Cas9 identification of a DNA sequence matches, it is from this chain start end inspection. Then, it will sequentially check the chain all the bases. For each nucleotide match, Cas9 will gain energy, and any mismatch will consume energy. A DNA sequence contains no matching of the bases more, and these mismatched bases closer to this sequence at the beginning, Cas9 less likely for it to be cut, on the contrary, it will from the DNA, fall down and continue looking for a better able to match its RNA template to the DNA sequence.

according to Depken of the argument, surprisingly, his team developed this simple mathematical models with very good prediction of the available information regarding the Cas9 cutting behavior of the data. If it does not match the nucleotide in a segment of DNA sequence at the end, then in order to increase the cutting possibilities, Cas9 may need to gathering enough energy to overcome this barrier. The model also explains Cas9 in a section of the DNA sequence of the startingAt the encounter does not match the base, or two mismatched bases close enough, why is it not on this piece of DNA sequence to be cut.

7. Angew Chem: a breakthrough! Will be moving the nanomotor makes CRISPR-Cas-9 got into the Cancer Center nest for gene editing!

doi:10.1002/anie. 201713082

in Cancer Research,“Cas-9–sgRNA”complex is an efficient gene editing tools, but it passes through the cell membrane in contact of the tumor cell genome of the capacity is very low. From the U.S. and Danish scientists have now developed a Can the movement of the nano-motor can effectively transport and release of this gene to the magic cut system. In this article published in the Angewandte Chemie article, the researchers describe in detail their development of ultrasound-driven Nano-motor.

genetic engineering is a promising cancer therapeutic tool in adaptive bacterial immune defense system“CRISPR”found that after experiencing a booming development. Currently used for gene editing of the engineered CRISPR system consists of two components: a single-stranded guide RNA（sgRNA and the Cas-9 nuclease,wherein the sgRNA can guide the Cas-9 nuclease binding specificity of the gene sequence, then the nucleic acid enzyme to exert its gene-editing capabilities. However the delivery of this huge gene-editing system into the cells in the presence of a certain problem. The research communications author Liangfang Zhang and Joseph Wang from the University of California, San Diego（UCSD）, they and colleagues have now developed an ultrasound-powered nanowire, can take the initiative to the Cas9–sgRNA complexes transport through the cell membrane into the cell.

gold nanowires can passively pass through the cell membrane, but due to their special asymmetric shape, the ultrasonic can promote their active movement.“ Gold Nano-wire the motor to the asymmetric shape for ultrasonic driving is essential.” The authors emphasize the know. They put the nano-motor with the Cas9–sgRNA complexes through the disulfide bond together. These may be the reduction of the chemical bond in the tumor cells has the advantage that they can be cancer cells within the rich natural reducing substances glutathione interrupted, which prompted the Cas9–sgRNA complex is released into the nucleus to play a role, such as the knockout gene.

as a test experiment, the researchers examined expression of green fluorescent protein in melanoma cells B16F10 in the fluorescence intensity change. He used ultrasound on cells treated for 5 minutes, which can be accelerated by carrying the Cas9–sgRNA complexes of the nano-motor through the cell membrane into the cell. They found the Cas9–sgRNA complexes can be in very low concentration effective to inhibit the fluorescent protein expression.

8. Nat Microbio: major discovery! Scientists found that the new CRISPR/Cas9 system!

doi:10.1038/s41564-017-0103-5

the CRISPR/Cas9 is a promising gene magic cut, can be of plant, animal and microbial genomes in particular DN a sequence for gene editing, can even be used to repair mutations. Recently, one by the University of Freiburg, Germany Juliane Behler and Wolfgang Hess Professor-LED research team discovered a new enzyme-one that relates to CRISPR/Cas9 systems as well as regulating normal gene expression of specific RNA scissors. Relevant research results published in Nature Microbiology.

natural CRISPR/Cas9 system found in most bacteria and archaea, which form a bacterial immune system to protect bacteria from virus damage. In order to protect the bacteria, a long RNA molecule will first be cut into many smaller units. Then an enzyme can use a pair of RNA scissors to cut RNA molecules to protect the bacteria. Natural the CRISPR/Cas9 system usually work on their own: they will be in different bacteria between the rapid exchange, while not dependent on a host of other proteins. Of course the existing popularity of the CRISPR/Cas9 system, there are exceptions, some systems will use the host enzyme RNA 3 as RNA scissors.

and from the University of Freiburg scientists have now found that RNase E can also be used as blue-green algae bacteria, the CRISPR/Cas9 system of RNA scissors. This enzyme is very common, not only in photosynthetic cyanobacteria bacteria, but also occur in some pathogenic bacteria and plant chloroplasts. It is all of these species the correct regulation of gene expression an important factor. But so far it is not clear whether it is in the CRISPR/Cas9 system may also play a role.

9. Genome Res: enhanced Edition CRISPR-Cas9: can more precisely edit the genes! Off-target effects is greatly reduced!

doi:10.1101/gr.226027.117

gene therapy is the treatment of genetic mutations leading to diseases of a new policy. A method of gene therapy involving the use of gene editing technology CRISPR-Cas9 directly repair the defective gene. Although it has therapeutic potential, but also will lead to unwanted and even harmful genetic errors, thus limiting its clinical application. In a study recently published in the Genome Research of a new study, from the Osaka University researchers have found an improved version of the CRISPR-Cas9 system, can significantly reduce editing errors the case for gene editing.

CRISPR-Cas9 system by the Cas9 protein to cut DNA, and a short guide RNA（sgRNA, telling Cas9 the cutting place with play effect. These two molecules together may be targeted to the genome in any gene editing. But the greater challenge lies in how to target to edit genes to achieve a particular gene changes.

“The problem is that Cas9 cutting is very difficult to precise the way of repair.” Nakada said.“ Therefore we use an improved version of Cas9, the only attack of a chain. We found that when we put the target gene and donor DNA after binding, we can more precisely fix the target gene.”

the researchers found that compared with the conventional technique, this technique can significantly reduce the accidental mutations. In one experiment, the traditional techniques in more than 90% of the cases will produce unexpected mutations, and that new technologies produce unexpected mutations in the case of less than 5%. It is worth noting that this precision technology and without sacrificing the gene editing efficiency.

10. Sci Adv: the blockbuster it! Scientists use CRISPR/Cas9 technology for successful treatment of Duchenne muscular dystrophy

doi:10.1126/sciadv. aap9004

recently, a study published in the international journal Science Advances on the study, from the United States and German scientists through the research describes a new CRISPR method, this new method may be expected to use of Duchenne muscular dystrophy（DMD）disease patients the body’s pluripotent stem cells to generate healthy heart muscle, this new method also overcomes the earlier researchers of the DMD cells for gene editing encountered a variety of problems.

since the disease is based on mutations induced, and therefore it may be able to become the CRISPR technology is successful treatment of the next plague, but the problem is the disease is often based on a large number of mutations nearly 3000 of the produce, in order to overcome this problem, researchers use a method called“myoediting”technique, in this technique, the CRISPR mainly will be targeting the role of mutations in hot spot region, which can be effective for the entire gene clusters for editing and repair.

In this study, the researchers also from DMD patients the body the body has developed induced pluripotent stem cells, then these cells are carried out specific repair and reprogramming so that it can grow into cardiac cells, and finally produce dystrophin; and by providing a plurality of cells to build a“scaffold”that the researchers will be able to produce a small amount of myocardial tissue, at the same time they also found that the generation of myocardial tissue not only capable of beating, also be due to the muscular dystrophy protein to produce and continuously maintain health. Biological Valley Bioon.com

Published at Sat, 03 Mar 2018 01:01:55 +0000