Crispr基因敲除细胞系系统分析-源井生物

Crispr knockout cell line system analysis-source well Bio

gene knockout cell lines of the prerequisites, is to a method for the elimination of certain tissues such as liver in particular gene technology. The technology for the study of individual genes in living organisms, the role is very broad. It with the traditional gene knockout technology is different, because it is at a specific time for a specific gene, rather than from the start it will be cleared. While Gene Knockout technology used to eliminate many of the traditional gene knockout side effects. In the conventional gene knockout, the embryos may be due to mutation and death, which make scientists can not study the adult gene cells. However, some of the cells of the tissue even if you can knock out also can not separate studies, so the cells in some organizations must be non-active, while in other tissues must be kept active. With this technology, scientists can be in the developmental stage-specific gene knockout cell lines, and studies of knockout an organization within the gene how it affects the other tissues of the same gene.

Easi-CRISPRfor use longssDNAthe donor to create knock-in and conditional knockout mouse model

based on CRISPR/Cas9 genome editing may be by destruction of the gene sequence to easily produce the knockout mice of the primary cells, but creation requires the insertion of exogenous DNA, knock-in, or replace a genomic fragment of the model efficiency is very low. This study used in most mouse models involve a knock-in（reporter gene or a recombinant enzyme or gene replacement, for example, contain a side wing of the LoxP sites in exon conditional knockout allele of. Has been reported to create this model some of the methods. These methods use double-stranded DNA as the donor, but the efficiency is usually 1 to 10%, and therefore unsuitable for General use. Researchers have recently shown that long single-stranded DNA（ssDNA）can be a very effective knockout donor, whether it is insertion or gene replacement. The researchers said this method to ssDNA insert-CRISPR（Easi-CRISPR effective Supplement because it is an effective technology, efficiency is typically 30-60%, and in some cases up to 100 per cent. The agreement will take about 2 months to produce founder mice, and thus studies from knockout mice primary cells and editing.

CRISPR-Cas9endonuclease generating a gene knockout cell lines of the condition, reveals coronary heart disease in Caenorhabditis elegans neural development the role of

gene knockout animal cell lines is the study of cell and Developmental Biology basic mechanisms a valuable tool. Researchers by manipulating the Caenorhabditis elegans somatic cell lineages in the RNA-guided DNA endonuclease CRISPR-Cas9 expression, we developed a conditional knockout strategy. The researchers showed that the CRISPR-Cas9 somatic technique provides a fast and effective way to in different developmental stages of various cell types to produce conditional gene knockout. In addition, the researchers demonstrated that this method is superior to our recent development of somatic TALEN technology, and can further generate a plurality of genes in cells of conditional knockout. By combining these techniques with live-cell imaging combined, the researchers showed that the human neural behavioral function have abnormalities associated with essential embryonic genes Coronin can be adjusted to neuroblastoma cell migration and neurogenesis during Central nematode actin organization. And cell morphology. The researchers suggest that the somatic CRISPR-Cas9 platform is particularly suitable for gene editing conditions for Biomedical Research.

the use of inducible telomere/masking proteinCRISPR/Cas9knock-out cells of the human telomere dysfunction tosystem analysis

the CRISPR/Cas9 gene knockout technology can use the human cells for human gene for effective loss of function analysis. However, the genes required for the study of the conditions required to knockout KO）cells. The researchers describe can be induced by CRISPR KO human cell lines to generate the telomere/shelterin complex. TRF1, AND TRF2, AND RAP1, AND TIN2, TPP1 and POT1 subunits and telomere interaction or can interact with other telomere separation. Several subunits of the homozygous inactivation in mice is lethal. On the human telomere regulation sub-function of the loss of most of the studies are dependent on the RNA interference-mediated gene knockout, which has its own limitations. We can induce CRISPR method allows us to more easily obtain a large number of cell lines, where required for telomere modulators has been inactivated, for biochemical and molecular studies. Our systems analysis revealed that human and mouse telomeres protein in the DNA Damage Response, telomere length and metabolic control in terms of functional differences, so as to maintain the telomere provides new insights.

Ubigene goal is to simplify genome editing. Ubigene developed CRISPR-U, based on CRISPR/Cas9 technology, and in double-strand breaks than ordinary CRISPR/Cas9 more efficiently, and CRISPR-U can greatly improve the efficiency of homologous recombination and easily achieve the knockout（the body）（in vitro point mutation PM, and a knock-in KI-in. By means of CRISPR-U, Ubigene have successfully edited more than 100 cell lines of the gene.

Reference

Miura H, Quadros R M, Gurumurthy C. B., et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors[J]. Nature Protocols, 2018, 13(1): 195-215.

Shen Z, Zhang X, Chai Y, et al. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. [J]. Developmental Cell, 2014, 30(5): 625-636.

Kim H, Li F, He Q, et al. Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells[J]. Cell discovery, 2017, 3(1).

gene knock in addition to the cutting efficiency can not only let a person have to generate protein glycosylation spectrum and the establishment of the regulation recording capacity, and has a variety of advantages, is a particularly attractive recombinant protein expression system. First of all, it is in the glutamic acid on the carboxyl group in tyrosine on the sulfation. Second, the operation is simple, by transient gene expression can rapidly produce recombinant proteins. Third, can be used for stable recombinant protein production. Some researchers use gene cells knock-out of the cutting efficiency of the system to produce gene edited cell lines, for GLUL genomic loci for targeted sequencing, to produce the EPO in stably transfected cell lines, and found that recombinant erythropoietin in the human body within the stable expression mechanisms.

according to research needs, the combination of target genes for gene stably transfected knockout programme design

scenario 1: a small fragment of the gene knockout program, the gRNA located in exon 2 at both ends of the intron, the knock except for the exon encoding the nucleotide number of non-3 ratio, the knock can be caused by shift code.

Scenario 2: frameshift gene knockout program, the gRNA located in the exon, the missing base number is for the non-3 multiples, knockout can occur frameshift mutation.

Scenario 3: a large fragment of the gene knockout program, the entire gene coding sequence knockout, to achieve a large fragment of the knockout effect.

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Published at Thu, 13 Aug 2020 18:55:43 +0000