CRISPR/Cas before the world in this life,you want to know are in there!

three

works

in a specific work process, the CRISPR sequence and Cas protein complexes, generally 3 steps to perform defense functions.

of exogenous DNA capture

to put it simply, the CRISPR/Cas system first to get a segment of exogenous DNA, to achieve the“Black List registration.” The CRISPR/Cas system will identify the intruder, such as viruses,“name”（PAM）and find it in the“ID”of the original interval sequence, and then the intruder identity information as a“profile”of a spacer sequence recorded to the“blacklist”of CRISPR sequences in. The following figure shows the��Stage of the works. When the phage virus for the first time invasion of bacteria, viruses, double-stranded DNA is injected into the cell interior. The CRISPR/Cas system from the period of the exogenous DNA in the interception of a sequence as the exogenous DNA of the“identity card”, and then as a new spacer sequence is integrated into the genome of the CRISPR sequence into. Therefore, this section and the spacer sequence corresponds to the“ID”is called before the interval sequence protospacer it. However, the“ID”of the selection is not random. The original interval sequence to the ends extending a few bases are very conservative, called before the interval sequence near the base sequencer protospacer adjacent motif, PAM on. PAM usually by NGG of three bases constituting the N is any nucleotide in. Virus invasion, Cas1 and Cas2 encode the protein will scan this segment of exogenous DNA, and to identify the PAM area, and then will be near the PAM DNA sequence as a candidate before the interval sequence. Subsequently, Cas1/2 protein complexes will be before the interval sequence from the exogenous DNA is cut down, and in the other enzyme with the assistance of the original interval sequence is inserted near the CRISPR sequence of the leader region downstream. Then, the DNA will be repaired, and will open the double-stranded gap is closed. As a result, a new spacer sequence was added to the genome of the CRISPR sequence into.

crRNA synthesis

the current study shows that the CRISPR/Cas system there are three ways Typeⅰ,ⅱ, ⅲ to synthetic crRNA, CRISPR/Cas9 system belongs to the Typeⅱ, currently most Mature and is the most widely used type. When viruses, the CRISPR sequence of the front guide area of the regulated transcription of the pre-CRISPR-derived RNA, pre-crRNA and trans-acting crRNA（tracrRNA it. Wherein, the tracrRNA is composed of repeated sequences areas transcription with the hairpin structure of the RNA, and the pre-crRNA is composed of the entire CRISPR sequence is transcribed into a large RNA molecule. Subsequently, the pre-crRNA and tracrRNA, and the Cas9 encoding the protein will be assembled into a composite body. It will be based on the intruder type, select the corresponding“ID”of the interval sequence of the RNA, and the RNase ⅲ （RNaseⅲ the assistance of this section,“photo identification”to cut, eventually forming a short small crRNA contains a single type of spacer sequence RNA as well as part of a repeating sequence of the area. crRNA and Cas9 and tracrRNA components of the final composite, as the next cut to be ready.

target to interference

as shown below, in the virus of the secondary infection, the Cas9/tracrRNA/crRNA complex can of the intruder’s DNA for precise strike. Complex scans the entire exogenous DNA sequence, and identified with the crRNA is complementary to the previous spacer sequence. In this case, the composite will be positioned to PAM/the pre interval sequence of the region, double-stranded DNA will be unlock. crRNA with the complementary strand, while the other chain remains in the Free State. Subsequently, the Cas9 protein to play a role, cut crRNA is complementary to the DNA strand and non-complementary DNA strand. In the end, Cas9 makes a double-strand break（DSB）formation, exogenous DNA expression is silent.

four

value

the CRISPR/Cas powerful that it can genes point the exact edit. In the Wizard RNA, guide RNA, or gRNA and the Cas9 protein interaction, cell genome DNA, as the exogenous DNA will be precisely cut. However, the CRISPR/Cas9 shear need to meet several conditions. First, to be edited near the area require the presence of a relatively conserved PAM sequence NGG-in. Second, the guide RNA with PAM upstream of the sequence complementary base pairing of. The most basic application is the gene knockout. If the gene downstream of each of the designed a guide RNA（gRNA1, the gRNA2 to its containing Cas9 protein-coding gene plasmid together into cells, the gRNA by complementary base pairing can be targeted PAM vicinity of the target sequence, the Cas9 protein makes the gene downstream of the DNA double-strand breaks. Then the organism itself in the presence of the DNA damage repair response mechanisms, will break the upstream and downstream ends of the sequence are connected, from the��To achieve a cell of the target gene knockout. If on this basis is a cell to introduce a repair template plasmid for the donor DNA molecules, that cell will be in accordance with the template provided in the repair process of the introduction of the fragment insert Knock-in or Site-Directed Mutagenesis site-specific mutagenesis to. So that you can achieve gene replacement or mutation. With further research, the CRISPR/Cas technology has been widely applied, in addition to Gene Knockout, gene replacement and other basic editing mode, it can also be used for gene activation, the disease model, and even gene therapy.

CRISPR: watch magic clip how to conquer a DNA

first, look at the once-sensational case: The Sun Yat-sen University researchers are using CRISPR-Cas9 correction of the human embryo in the mutant. Chinese scientists use CRISPR/Cas9 to the human embryos will lead to thalassemia the β-globin gene mutations successfully be modified, the purpose is hoping to use CRISPR for such mutations to be corrected, in order to achieve the use of gene therapy for thalassemia. Articles published in the protein·cells of Protein & Cell journal on[1]. span>

beta-thalassemia is a potentially life-threatening blood disease, it is in the world the prevalence is about one hundred thousandth of. The researchers from the use of a β-thalassemia patient’s tissue to construct cloned embryos, and then these embryos in the DNA sequenced, the discovery of a single nucleotide error, i.e., it should be for A nucleotide to be replaced in order to G. Next, they research use the CRISPR such Bases Conversion back, i.e. to achieve a G-to-A. This is also the first confirmed use of the CRISPR system to cure human embryos in genetic diseases is feasible. But this study caused by the impact force of the reasons are: it is in the baby embryo inside carry out, though the doctor dropped is not successfully bred out of the baby’s abnormal embryos, but this point is still subject to some of the scholars of criticism and attack.

in the not long after, the New England Journal of Medicine on the NEJM reports the University of Basel researchers use CRISPR find the cause of polycythemia the first genetic mutation. [2]. span>

by using a whole-genome linkage analysis and gene sequencing, the researchers found that all affected family members of the EPO gene are missing a single BP. While the EPO increase is what causes the red blood cells too much causes. But confusing is that this base deletion causes a gene encoding reading frame movement occurs, eventually leading to the EPO gene loss of function instead of enhancement. But the reality is that a patient’s blood EPO content was increased rather than reduced. In the end, or CRISPR to help researchers find the answer. It turned out that the EPO gene, there is a hidden mRNA, the normal case does not participate in the formation of EPO. Mutations result in the gene’s reading frame from moving, causing this gene to produce more EPO.

in addition to in the blood aspects of the application, the CRISPR in the developmental aspects Zhuo has achievements. The recent “Nature” （Nature）it is reported that the British and South Korean researchers used CRISPR/Cas9 to reveal the OCT4 gene in human embryonic early development plays a key role. In the normal case, the OCT4 gene in the human embryo the first few days of development is active, it drives the fertilized egg split, about 7 days after formation of an about 200 cells consisting of a sphere, i.e., the balloon embryo. In the experiment, they used CRISPR/Cas9 to block human embryonic OCT4 expression after these the development of the embryo stopped[3]. span>

CRISPR another value that can be used to track cells, the function in cancer has an important role. Stanford University researchers on the CRISPR gene editing technology with DNA barcode technology combined with effective tracking of cancer progression.

human cancer is not only a tumor suppressor mutations, which exist in a variety of mutation combinations. In order to understand the different mutated gene is how the interaction, the researchers spent several years of efforts to draw the map, including building a variety of different lineages in the genetic modification of mice, each of which carry different inactivation of tumor suppressor genes. And if you want to explore all possible���Combination, the researchers will need thousands of mice.

CRISPR-Cas9 a powerful that can easily replace, modify or remove organisms within the gene sequence, resulting in individual mice of the lungs to create a variety of genetically different tumors. The problem is that, in order to draw conclusions about the different mutations combined effect of the useful conclusions, scientists need an accurate way to mark the track of different tumor growth. But if the CRISPR-Cas9 and DNA barcode technology the use of organisms DNA in a segment of conserved fragments of the species for rapid and accurate identification technique is combined in order to track the growth of cancer developments, it could help scientists in the lab to replicate the cancer patient in the body of the observed genetic diversity. Such as short, unique DNA sequence of the DNA barcode）adhesion in the mouse lung within a single tumor cells, so that each sequence of functions is like a genetic bar code, when each of the cancer cells amplification, the barcode number will increase. Finally just need the entire cancerous lung removed, and then used high-throughput DNA sequencing and computational analysis of the barcode frequency of occurrence, thereby accurately determining the tumor size.

in other words, researchers can be in the same mice produce a lot of having specific genetic characteristics of the tumor, and in the scale and accuracy are tracking their growth. The researchers only end in a few months on the completion of the relevant experiments, only took less than 24 mice. The results, published in the Nature Genetics on Nature Genetics on[4]. span>

similarly, Nature heavyweight sub-Journal of Nature·Biotechnology of Nature Biotechnology also reported that German researchers used CRISPR/Cas9-induced DNA scar sequence tracing cell lineages[5]. span>

in the biological world there has been a question stuck with everyone, the function of the different cell lines in the end from where? While CRISPR has a feature is always at the exact point on the cut, inspired by this, the German team of scientists developed what is known as LINNAEUS（lineage tracing by nuclease-activated editing of ubiquitoussequence, through the universal presence of the sequence of the nucleic acid enzyme activated in the edit to carry out lineage tracing technology, which would allow people to determine the cell type and each cell lineages.

in zebrafish embryonic cells how to cut DNA, in the next cell division occurs prior to DNA repair time does not exceed 15 minutes. Repair work must be done quickly, this is also the error accumulation of the place, this error sequence is called DNA scar sequence. The DNA in the scar sequence has a random length, and their exact position will change. Daughter cells during cell division genetic these scar sequence. When these scar sequence together when it’s like a barcode as the role can be determined for each cell lineages.

so the researchers designed an experiment in zebrafish embryos injection of CRISPR-Cas9 system, the zebrafish prior to be transferred to the red fluorescent protein（RFP）。 In the next period of time, the Cas9 repetitive cutting zebrafish in vivo RFP. These zebrafish embryos in the red fluorescence gradually disappears at the same time, thousands of scar sequences in the cell DNA damage region is formed. Ultimately, through the analysis of the scar sequence, we can determine the cells from which progenitor cells.

in summary, the CRISPR DNA editing plays an important role, also derived most of the new technology.

when a CRISPR case on the CAR-T: look at the intensity as strong as any join forces to overcome cancer

common type of CAR-T

first let’s talk about universal type CAR-T. Currently, the market most of CAR-T cells using patient’s own T cells to produce. This process is not only time-consuming, expensive, and also limited by current manufacturing capabilities. If scientists were able to think of a way to generate common type of CAR-T cells, and that this type of therapy will be expected to become more convenient and cheaper. Because these ready-made off-the-shelf cells will increase the ability to accept a single CAR-T cell products in the treatment of the patient number.

https://www.nature.com/articles/nrd.2016.238

but there are two problems become common type of CAR-T of the main obstacles,Graft-versus-host diseases, graft-versus-host disease, GVHD, and host rejection. Fortunately, the United States Memorial Sloan-Kettering Cancer Center scientists led the research team found that allogeneic donor CD19-specific CAR-T cells in killing tumor cells at the same time, the cause of the GVHD occurs the risk is minimal. Article published in Nature Medicine on on.

https://www.nature.com/articles/nm.4258

based on such a natural advantage, the researchers just want to use CRISPR to CAR-T gene-edited, reduced and CAR-T antigenicity. CAR-T pioneer Carl June, Professor in the Clinical Cancer Research Journal published article confirmed that in vitro and animal model studies, with CIRSPR knockout TCR-T cell receptor and B2M（β-2 microglobulin this two-and-immune exclusion-related genes after T cell allogeneic reaction of alloreactivity reduced, and did not lead to GVHD.

http://clincancerres.aacrjournals.org/content/23/9/2255.long it.

similarly, China and the UK scientists found that with CRISPR knock-out TRAC T cell receptor alpha constant chain after can construct the General type of CAR-T.

https://www.ncbi.nlm.nih.gov/pubmed/29605708

https://www.nature.com/articles/cr2016142

the author hereby would also like to add is that the General type of CAR-T there is another interpretation, that is, the same T cells can be loaded with different antigens. This concept is by MIT and Boston University scientists put forward. As shown below in T cells expressing the common-type receptor, and then this General type of receptor can be connected to different target antigens, the development of a separate, General-purpose, programmable type CAR system used for T cell therapy, they called for SUPER CAR-T. That is, the patients suffering from different cancer patients just need to input the same kind of SUPER CAR-T, and then enter the appropriate antigen such as lung cancer specific antigen, or lymphoma-specific antigens, etc., which greatly speeds up the treatment process.

https://www.cell.com/cell/fulltext/S0092-8674(18)30362-3

enhanced CAR-T

CRISPR in addition to the means for generating a generic-type of CAR-T, but also through the knock-out immune co-inhibitory pathways or signal molecules genes, such as CTLA4, PD1）to improve the CAR-T cell effect. More representative is the NIH the US National Institutes of Health）subordinates Recombinant DNA Advisory Committee approved by Carl June, Professor in leadership of a CRISPR clinical trials. In this test, the researchers will use CRISPR/Cas9 in the targeting of melanoma of the CAR-T in the knockout encoding PD-1 gene and the endogenous T cell receptor genes. This study by the University of Pennsylvania and its partners—the University of California, San Francisco, and MD Anderson Cancer Center co-responsible. The Nature Biotechnology magazine also specifically reported that the research team plans in a Phase I clinical trial results. Knockout PD-1, the results show that CAR-T killer stronger.

https://www.nature.com/articles/nbt0117-3

but the inhibitory PD-1 pathway is not a panacea, the Nature magazine reported that if in T-cell non-Hodgkin lymphoma(T-NHL)is cancer in a mouse model using PD-1 inhibitors immunotherapy, not only did not play a role in cancer, but also promote tumor progression. This result is actually expected, since in T-NHL, T cell is a tumor cell, inhibition of the PD-1 pathway will only accelerate tumor growth. Since PD-1/PD-L1 no, that CAR-T right? Then appeared another problem: the CAR-T cells and cancer T cells between the target antigen co-expression would lead to CAR-T cell auto-destruction. These problems are limited to the CAR-T cell therapy development.

but don’t worry, from Washington University in St. Louis School of Medicine scientists have used gene editing technique CRISPR to human T-cell transformation, in addition to the CD7 and the T-cell receptor alpha chain(TRAC)expression, which is a for the CD7+ T-cell malignancies, to avoid the“cannibalism”of the CAR-T cell therapy(UCART7)。 It can protect the normal T cells of the cases, the cancerous T cells initiate the attack. Research results recently���Table in an authoritative academic journal Nature magazine sub Journal of the Leukemia of the above, the results show: acceptance of gene edited engineered to CD7 to target the T cell treatment groups of mice, the median survival period of 65 days, and accept the control group of mice the median survival period of only 31 days.

https://www.nature.com/articles/s41375-018-0065-5

to summarize, CRISPR joint CAR-T can indeed be to play 1+1 > 2 Effect, together with the strong join forces to eliminate cancer. The hope for the future to have more breakthrough occurs.

in-depth analysis of the CRISPR clinical applications, and market Outlook

a

clinical applications

say to clinical application, Chinese scientists in this regard and come out on top. Although Zhang Feng, Jennifer Doudna and Carl June and other large cattle have been scrambling to apply a clinical trial, but this field pattern may have to be a number of“obscurity”of Chinese scientists to rewrite it! In 2016 7 months, theNatureannounced that the hospital Review Board ethical approval, the Sichuan University West China hospital oncologists Lu uranium the Professor will conduct the world’s first CRISPR human trials, the use of CRISPR technology to edit the T cells to the therapeutic chemotherapy, radiotherapy and other therapy ineffective treatment of metastatic non-small cell lung cancer patients with this clinical trial. In fact, China in the CRISPR field has been the action is very fast, has created a plurality of the first, as the first CRISPR to edit the human embryo, the first CRISPR to edit the monkey.

test the specific operation process is, firstly from the patient’s blood is extracted T cells, and then use the CRISPR/Cas9 technology to knock out the PD-1 gene. The gene is the body’s immune response to the key switch, remove it can restore T cell anti-tumor capabilities. By the CRISPR editing after the T cell in the laboratory after amplification, and then reinfusion into the patient’s blood. When the engineered T cells in the circulation to the lung tissue, can be not affected by the inhibition of tumor cell and destroy it. This scheme is also a worthwhile improvement, that is, the study group can be selected from tumor sections for T cells because these T cells have been ready to specifically attack cancer.

of course, in addition to China, other countries in this area put a lot of effort. In addition to the previous article“when the CRISPR-meets-CAR-T: look at the intensity as strong as any join forces to overcome cancer”mentioned Carl June lead to the use of CRISPR/Cas9 in the CAR-T in the knockout encoding PD-1 gene and the endogenous T cell receptor gene studies outside the United States CRISPR Therapeutics company first won the European regulatory Agency permission for conducting clinical trials the company will actively carry out the use of CRISPR to repair hereditary blood disease β-thalassemia patients of the defective gene.

the company this year 4 month has been to the FDA submitted a sickle cell anemia disease treatment drug CTX001 of the clinical trial application. CTX001 is CRISPR Therapeutics, and Vertex Pharmaceuticals co-development, co-commercialization of a study of in vitro gene editing therapies. Its by editing the patient’s stem cell genes, to increase the red blood cells of fetal hemoglobin（HbF levels, in order to achieve β-thalassemia and sickle cell disease treatment. HbF is a baby born with naturally occurring oxygen-carrying hemoglobin of a form, and later gradually is the adult form of hemoglobin instead. By elevated HbF CTX001 might alleviate β-thalassemia patients blood transfusion requirements as well as sickle cell patients with pain and debilitating symptoms. Although because of the review of the reasons the FDA moratorium on CTX001 of the investigational new drug application IND, but in a European clinical Phase I and phase ⅱ trial plan remains unchanged, is expected in 2018 in the second half of the start of the test.

in addition, Editas company will also be carried out for hereditary retinal diseases: 10 type of congenital Leber’s Black Simon（Lebercongenital amaurosis type 10, the biological interpretation of the Phase I trials, this study will use the gRNA-Cas9 nucleases, to correct the encoding centrosome protein 290（CEP290’s mutations.

two

market analysis

turning to CRISPR the market, you can use the hot is the word to describe. Not long ago, Feng Zhang���David Liu co-founded of the Beam The rapeutics successful refresh, you can reflect the market’s fiery degrees. The author here don’t want to repeat CRISPR market value, but rather to objectively analyze the CRISPR side effects.

first, attract everyone Eye is certainly CRISPR off-target effects, it is wanted to edit the target gene, the results of the“bullet”deviation, and edit an unrelated gene. The most representative is the Nature Methods article[1]claiming to be from Columbia University and other institutions of the researchers found that, in the cure of mice-related genetic disorders at the same time, gene editing commonly used tool CRISPR/Cas9 could cause a lot of unexpected mutations. The study, published once caused a large wave of academics on the CRISPR technique of criticism and accusations, and the impact of a number of ongoing based on the CRISPR gene editing technology in clinical trials.

but soon to be other scientists to refute, they said the study in the methods and logic on the presence of defects, including the number of samples is too small, the conclusions and data inconsistent with, etc. As the CRISPR leader in the field of characters, Editas Medicine Zhang Feng team and Intellia Therapeutics Jennifer Doudna team of scientists at the time were to write to the Nature Methods editorial, that the papers concluding the presence of bias, and demanded the papers the withdrawal of the draft.

face questioning, the Nature Methods of Investigation, and ultimately make the withdrawal of the draft decision. It is given the withdrawal of the draft on the grounds that article, the use of mice, although derived from the same“ancestor”, however, the gene editing mice and control mice also are not Pro-siblings relationship, even the Pro-siblings, the genome itself is the presence of the mutation may also have many different. The genetic background of the source is not clear, this creates a huge problem: these mice using CRISPR gene editing technology before, its genome would have existed many mutations, and non-gene editing after caused. Secondly, other research team through the analysis, there is no evidence that the CRISPR gene editing will then cause mutations. In order to give this field argue that to draw a perfect full stop, the Nature Methods on on 3 on 30, published from the gene editing field of dozens of top scholars and institutions in many articles in the newsletter articles, respond to this article.

a wave is not flat, after wave again. Just recently, two articles published in the Nature Medicine article[2,3]once again targeting CRISPR, the media reported that“the use of CRISPR-Cas9 successful editing of pluripotent stem cells is likely to have a p53 defect, which will increase cancer risk”,“these gene editing cells it is like a time bomb the same”. But this time, I have to emphasize that we have for these two articles were the correct interpretation.

in this case, the author would like to quote what the intellectuals of this event is to reply to the topic of the Article[4]。 In the first Article[2], the authors concluded that the CRISPR-Cas9 will trigger a p53-mediated DNA damage repair mechanisms, which makes the gene editing becomes more difficult, and if the artificial removal of the p53 gene, the lifting of this repair mechanism would allow cells to become more easily be edited. And in the second article[3], the authors feel, if p53 function is sound, the majority of cells will be editing fail. According to their estimates, in pluripotent cells, the p53 so that the CRISPR efficiency is reduced by 17 times as much. So in fact, these two articles concluded that p53-defective cells are more likely to be edited, and is not the cell to be edited after more likely to cause p53 defects. It cannot be confirmed that CRISPR has carcinogenic risk.

of course, the two articles raises a concern of the imaginary: the CRISPR won’t tend to edit containing the p53-deficient cells, and the loss of this“gene-keepers”of cells will have a high risk of cancer. But many experts believe that the p53 defective cells are more likely to be edited does not necessarily mean that after editing the cells more prone to p53 defects. CRISPR just leads to DNA double-strand breaks in one of two ways, using CRISPR to generate DNA breaks, is not than other manner to produce a fracture special, not particular to affect the p53.

thus, the real gold not afraid of fire to refining it. Any technology is not perfect, and CRISPR is no exception. But CRISPR genuine gene encoding��Efficiency, people have to imagine its limitless future.

lines so far, with regard to the CRISPR in a series of stories will come to an end, I hope everyone can have the harvest.