Release date: 2018-03-22 Gene editing technology has continued to evolve and has now evolved into the third generation of gene editing technology. The third generation gene technology CRISPR/Cas overcomes the shortcomings of traditional gene manipulation, such as long cycle, low efficiency and narrow application. As an emerging genome editing tool, CRISPR/Cas is able to perform RNA-directed DNA recognition and editing. The sequence-specific guide RNA is used to guide the endonuclease to the target sequence, thereby completing the precise editing of the genome. Because of its simple operation, low cost and high efficiency, it has become a hot gene in recent years. Editing methods have been widely used in model biology research, medical, plant crops, agricultural animal husbandry and other fields. 1. Sharpen the sharp weapon to speed up scientific discovery The emergence of CRISPR/Cas9 gives scientists the possibility to imagine. The CRISPR/Cas9-based technology is quickly being used in various laboratories around the world. Here we will mainly introduce the most commonly used applications. In the early days, researchers used gene-targeting techniques mediated by homologous recombination (HR) to achieve gene editing, but their efficiency was too low, which greatly limited their application. To overcome this problem, a series of endonuclease-mediated gene editing techniques have been developed to cleave specific genomic sequences by these endonucleases, using cell-based repair systems such as non-homologous end joining or homologous recombination. The repair method, and thus the purpose of changing the genomic sequence, zinc finger endonuclease (ZFNs), transcription activator-like effector nucleases (TALENs), and sgRNA-mediated Cas9 endonuclease work based on this principle. of. Zinc finger endonucleases (ZFNs) and transcription-like activator effector nucleases (TALENs) can recognize specific DNA sequences on the genome and perform cleavage at specific sites through protein-DNA interactions, but they are inefficient, The limited potential sites and high cost have greatly limited their application. Until the emergence of the CRISPR/Cas9 system, researchers have found a low-cost, efficient and easy-to-use gene editing tool. After the emergence of CRISPR/Cas9, the first thing scientists think of is to apply it to gene editing. Design single guide RNA (sgRNA) according to the exon sequence of the target gene and transfer it to the cell together with the plasmid containing the Cas9 coding sequence. sgRNA guides the Cas9 protein to target DNA sequence through the principle of base-pair pairing. The Cas9 protein cleaves DNA at this site, triggering DNA double-strand break (DSB), at which time the cell is repaired by non-homologous end joining (NHEJ) Complete self-repair of DNA, due to the frequent addition and loss of bases during the repair process, and eventually result in frameshift mutation of the gene to achieve the purpose of gene knockout, or design an sgRNA for each of the upstream and downstream sequences of the target gene, thereby triggering DSB occurs simultaneously in the upstream and downstream of the gene, and the DNA at the upstream and downstream ends of the cleavage is linked together by a DNA damage repair mechanism to induce DNA fragment deletion, thereby achieving the purpose of gene knockout. If a repaired template plasmid is introduced into the cell based on this, the cell will be homologously recombined with this template. If the introduced repair template is a gene to be inserted, the gene can be knocked in at a specific position. . 2, the magic dCas9 multi-function kit With the deepening of Cas9 research, the structural basis of Cas9's function is gradually becoming clear. When Cas9 plays the role of cutting DNA, its two domains play an important role, namely RuvC and HNH, of which HNH structure is responsible. The cleavage of the sgRNA complementary strand, the cleavage site is located outside the third base of the 5' end of the PAM, and the RuvC domain is responsible for the cleavage of the non-complementary strand, which is between 3-8 bases upstream of the PAM. Inactivation of both of these mutations resulted in the Cas9 protein (dCas9), which lost its DNA cleavage activity. Although dCas9 lost its ability to cleave DNA, it could still reach the designated DNA sequence under the guidance of sgRNA. Based on this feature of the sgRNA–dCas9 complex, if the different functional domains are fused on dCas9, different modifications can be made in specific DNA regions, which forms a CRISPR/dCas9-based toolkit. Scientists with brain-opening use dCas9 protein to develop tools for various purposes, which can be used to make the use of CRISPR/dCas9. Here are a few simple examples, such as the researchers designing sgRNA for the promoter sequence of the target gene. The sgRNA–dCas9 complex binds to the promoter of the target gene, and the steric hindrance brought about by the dCas9 protein can interfere with the binding of the transcription factor, thereby triggering the effect of interfering gene expression at the transcriptional level, on the basis of which In order to achieve better interference effects, some domains capable of triggering gene transcriptional repression are also fused to dCas9 protein, such as KRAB (Krüppel-associated box). Since gene expression interference can be achieved by CRISPR/dCas9, can it also activate gene expression through CRISPR/dCas9? The answer is yes. Researchers have promoted the endogenous activation of genes by merging vp64 (four tandem vp16) and p65AD (p65 activation domain) onto dCas9 to achieve endogenous activation of genes. After various optimizations, such as vp64, The triple domain consisting of p65AD and VPR (Epstein-Barr virus R transactivator Rta47) (dCas9–VPR) can achieve high levels of endogenous activation of gene expression. Through the establishment of CRISPR/dCas9-based gene expression interference and endogenous activation tools, researchers have a simpler, more efficient, and lower-cost research tool for conducting work such as gene function research. This largely saves researchers time and money. 3, precise editing of epigenetic modification Epigenetic research is a very hot field in recent years. DNA methylation and histone acetylation play important biological functions in organisms, and CRISPR/dCas9 has become an epigenetic study. Very powerful tool. For example, CRISPR/dCas9-mediated targeting of DNA methylation, we know that DNA methyltransferases (DNMTs) play a key catalytic role in DNA methylation, and most of the DNA methylation Occurred on the CpG island, so the researchers tried to fuse the catalytic domain of DNMTs to dCas9 to form dCas9-Dnmt3a3L, and catalyze methylation of CpG near the target DNA sequence by sgRNA guidance to achieve DNA methylation. Fixed point editing. Similarly, the researchers fused the catalytic domain of the TET1 protein, which plays a key catalytic role in DNA demethylation, to dCas9 to form dCas9-TET1, which is also targeted by the sgRNA to target CpG near the target DNA sequence. Demethylation modification is achieved. Like CRISPR/dCas9-mediated targeting of histone modifications, similar to targeted DNA methylation modifications, some of the enzymes involved in histone modification include histone demethylase (LSD1/KDM1A), histone acetyltransferase Enzymes as well as histone methyltransferases and the like are also fused to the dCas9 protein to achieve targeted histone modification. In addition to the above applications, CRISPR/dCas9 has also been used in many other fields, such as the fusion of EGFP to dCas9, and targeting of specific DNA sequences by sgRNA to achieve gene composition images. In addition, researchers have developed CRISPR/dCas9-based enChIP technology to detect DNA-protein interactions on specific genomic regions, immunoprecipitation of antibodies labeled with dCas9 targeting specific genomic loci via sgRNA, followed by protein profiling ( enChIP-MS), identifying proteins that interact specifically with it. The development of these tools has greatly helped researchers to make operations that were previously impossible, and promote the rapid development of life sciences. 4. Determination of gene and drug target genes In the past, ZFN or TALENs-based genome editing technology required designing proteins for DNA target sequences. The CRISPR technology only needs to synthesize corresponding 80 nt sgRNAs according to different target sequences to guide the Cas9 protein to modify the sequence, which enables gene editing. High-throughput applications of technology. CRISPR genome-wide screening technology can be used to identify essential genes and drug target genes. The University of Toronto's Jason Moffa team established a "core fitness genes" that covered the genome-wide gRNA library and knocked out 18,000 genes one by one in five cell lines, and finally identified 1,580 essential genes that were conserved across different cell lines. Similarly, the W. Nick Haining team at the Dana-Farber Cancer Institute in the United States systematically knocked out 2368 genes of melanoma cells by CRISPR/Cas9, and found that deletion of the ptpn2 gene would make these cancer cells block PD-1 more. sensitive. The Michael Diamond team at the University of Washington School of Medicine used CRISPR/Cas9 to identify nine genes that are absolutely essential for the infection of flaviviruses in host cells. The absence of the spcs1 gene not only reduces the rate of flavivirus infection, but also does not cause side effects on cells. This will be a potential target for flavivirus drugs. 5. Disease modeling and organ donor production As a next-generation gene editing technology, CRISPR/Cas9 can also be used to establish disease models and to cultivate donor organs. Gene therapy can correct genetic defects in patients' own cells, and combines other biological techniques to develop tissue-specific "organs" in vitro, which is of great significance for disease modeling, drug screening and clinical treatment. . The CRISPR-mediated genome editing technology can be directly applied to the establishment of disease models in non-human mammals, which will be more conducive to disease pathogenesis and cure research. In addition, CRISPR technology can also be applied to genetic editing of large animals to study immune rejection and cross-species disease infection, thus solving the bottleneck of xenotransplantation source. Pig is considered to be the preferred animal of human heterogeneous organ source, and currently used for pig organs. The main obstacles in humans are immune rejection and the medical risks associated with Porcine endogenous retroviruses (PERVs). Dr. Yang Wei from eGenesis and Professor George Church from Harvard University used CRISPR for genetic modification to shut down 62 PERV pol genes, thus reducing the risk of infection from PERV by three orders of magnitude and successfully breeding pig strains without PERVs as safe and effective. The source of xenograft organs, these studies make pigs a more promising source of organ for patients. 6, gene therapy Gene editing technology can accurately transform human genes to achieve gene therapy effects. Li Jinsong, from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, corrected the genetic defects in a mouse model of cataract by injecting CRISPR/Cas9 into mouse embryos. The resulting offspring are fertile and can correct the alleles. Passed to their descendants. Duchenne muscular dystrophy (DMD) is a rare form of muscular dystrophy and one of the most common fatal genetic diseases caused by mutations in the dystrophin gene. Duke University's Charles Gersbach team used CRISPR/Cas9 to cleave the 23 exon of the dystrophin gene mutation in DMD mice, and synthesized a truncated but highly functional dystrophin, a biologist. “The first successful use of CRISPR gene editing technology to cure a genetic disease in an adult living mammalâ€. Gene editing technology combined with immunotherapy has broad application prospects in cancer and HIV/AIDS treatment. Chimeric Antigen Receptor T cell (CAR-T) cell therapy is a very promising approach to tumor therapy. CAR-T cell therapy has achieved great results in the treatment of B cell malignant hematological tumors. The research team of the Institute of Zoology of the Chinese Academy of Sciences, Wang Yiyi, used CRISPR/Cas9 technology to perform double gene (TCRα subunit constant and beta-2 microglobulin) or three genes (TRAC, B2M and programmed death-1) knockout in CAR-T cells. The Michel Sadelain team at the Sloan Kettering Cancer Memorial Center in the United States found that CRISPR/Cas9 technology specifically targets the insertion of CAR genes into the TRAC locus of cells, greatly enhancing T cell potency, and editing cells are much better than traditional in acute CAR-T cells are produced in a mouse model of lymphocytic leukemia. Following the launch of Novartis' Kymriah and Gilead (kite Pharma)'s Yescarta, CRISPR Therapeutics is also developing CRISPR/Cas9 gene editing technology to develop allogeneic CAR-T candidates. In October 2016, a team led by Lu Yu, an oncologist at West China Hospital of Sichuan University, first conducted a CRISPR test in humans, extracting immune cells from patients with advanced non-small cell lung cancer, and then using the CRISPR/Cas9 technology to eliminate PD in cells. The -1 gene is more helpful in activating T cells to attack tumor cells, and finally re-injecting the genetically edited cells into the patient. 7, pathogenic bacteria and anti-virus research Microbial populations are closely related to human medicine and the natural environment. The University of North Carolina Rodolphe Barrangou and Chase L. Beisel collaborate to target and distinguish highly closely related microorganisms and procedurally remove bacterial strains by using the genomic-targeted CRISPR/Cas9 system, meaning that the CRISPR/Cas9 system can be developed into elaborate microbial treatments. The system will eliminate harmful pathogens, and humans will be able to precisely control the composition of the microbial population. Udi Qimron of Tel Aviv University in Israel introduced the CRISPR system into mild phage to infect antibiotic-resistant bacteria to eliminate such bacteria. The CRISPR system has the potential to become a new class of antibiotics. Locus BioSciences is also developing the development of a CRISPR system in bacteriophage for the purpose of eradicating Clostridium difficile. Virginia Tech's Zhijian Tu research team edited the M factor gene in male mosquitoes, which can lead to the conversion between male and female mosquitoes or the killing of female mosquitoes, thus achieving effective sex separation and effectively reducing the number of mosquitoes, and also reducing Zika virus. And malaria and other spreads. Based on CRISPR therapy, it can be applied not only to pathogens that eradicate commensal or beneficial bacteria, but also to human viruses, including HIV-1, herpes virus, papillomavirus and hepatitis B virus. Patients with a homozygous 32-bp deletion (Δ32) of the CC chemokine receptor type 5 (CCR5) gene are resistant to HIV infection. Therefore, the University of California, Yuet Wai Kan, induced the differentiation of monocytes and macrophages to be resistant to HIV infection by introducing a homozygous CCR5Δ32 mutation in the induced pluripotent stem cell iPSC using the CRISPR system. The Kamel Khalili team at Temple University used the CRISPR/Cas9 system to accurately edit the HIV-1 LTR U3 region in the host cell genome, thereby removing HIV from the genome. 8, nucleic acid diagnosis and cell event record Cas12a (Cpf1) belongs to the CRISPR family, another endonuclease that can also be directed by gRNA and cleave DNA. However, it can not only cut the single-stranded or double-stranded DNA but also cut other DNA. Recently, the Jennifer Doudna research team at the University of California at Berkeley developed a new CRISPR-based genetic detection (DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR). Using single-stranded DNA to link fluorescent molecules and quenching molecules into a report In the system, when CRISPR-Cas12a binds to the target DNA under gRNA guidance and acts as a cleavage, the DNA in the reporter system is also cleaved, and the fluorescent molecule will be unsuppressed. This system detects DNA samples in humans with oncogenic HPV. HPV16 and HPV18 are excellent. The CRISPR-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) developed by the Feng Zhang research group of the Broad Institute, the principle is that after Cas13a is activated, it can cut the characteristics of other RNAs other than the target sequence, and introduces the fluorescence release. Molecular inhibition. This tool enables one-time multiplex nucleic acid detection, which can detect four target molecules simultaneously. The additional Csm6 makes this tool more sensitive than its predecessor, and developed it into a micro test strip detection method, which is simple and clear. Easy to operate, it has been successfully applied by researchers to RNA viruses such as dengue virus and Zika virus, as well as human body fluid samples. The Broad Research Institute's David R. Liu team used CRISPR/Cas9 to develop a "black box" for recording cellular events called the CAMERA (CRISPR-mediated analog multi-event recording apparatus). They used the system to develop two cell records. System, in the first cell recording system called "CAMERA 1", the researchers used the self-replication of the plasmid in the bacteria but strictly controlled its own number of features, and the two plasmids that were slightly different from each other were A stable ratio was converted to bacteria, and then one of the two plasmids was cleaved using CRISPR/Cas9 upon exposure to foreign drug stimulation, and bacterial contact was recorded by sequencing the plasmid and recording changes in the ratio of the two plasmids. The time of external stimulation. Another cell recording system, called "CAMERA 2", uses a CRISPR/Cas9-based base editing system to change a single base in a genetic sequence when a specific signal occurs in a cell, thereby achieving Contact with stimuli such as nutrients. The emergence of this technology will greatly help people to understand more about the occurrence and development of various life activities of cells. 9, human embryo genome editing In April 2015, Huang Jun of Sun Yat-sen University used CRISPR/Cas9-mediated gene editing technology to restore a mutation in the embryo that causes β-globin gene (HBB) in the embryo. In 2016, Fan Yong team of Guangzhou Medical University in the three original nuclear fertilized eggs, using the gene editing technology CRISPR gene in the fertilized egg CCR5 for editing to introduce CCR5Δ32 homozygous mutations due to the problem of off-target efficiency at that time, resulting in mosaic fertilized eggs. On August 2, 2017, the Shoukhrat Mitalipov team at the Embryo Cell and Gene Therapy Center of the Oregon Health and Science University published the results of DNA editing using CRISPR in human embryos, correcting the mutated MYBPC3 gene, which causes cardiac hypertrophy. And the young athletes will die. Source: billion euros Delicious Fresh Frozen Octopus delicious Fresh Frozen Octopus Delicious Octopus,Delicious Frozen Octopus,Delicious Fresh Frozen Octopus,Delicious Frozen Fresh Octopus Zhoushan Junwei Aquatic Products Co., Ltd. , https://www.junweiaquatic-intl.com