Latest research progress in gene knockout technology-|gene knockout technology|transgenic research|gene cloning|gene targeting operation|
Gene knockout
The technology is a molecular biology technology developed on the basis of gene homologous recombination technology and embryonic stem cell technology. In 1987 Thompsson established a complete mouse model of ES cell knockout. In recent years, there have been emerging gene knockout technologies based on high-efficiency targeted modification and regulation technologies of ZFNs, TALENS, and Cas9. In January 2012, the "Nature Methods" magazine of the genome editing nuclease technology was selected as the annual research method. In December 2012, it was named one of the top ten scientific advances in 2012 by Science magazine. These gene knockout techniques have greatly improved the efficiency of gene knockout and have a very high knockout specificity, which has created a new way to study gene function and will greatly promote the development of biology and medical research. The commonly used gene knockout techniques mainly include the following categories.
1 Traditional gene knockout method
1.1 Gene knockout using homologous recombination
Gene knockout, also known as gene targeting, is a genetic engineering technique that involves DNA homologous recombination between a foreign target gene and a nuclear genome target gene in a transfected cell, enabling the integration of the foreign gene into the nucleus. The specific location of the genome, thereby achieving the goal of altering the genetic characteristics of the cell by gene knockout. Traditional gene knockout technology relies on the random exchange of naturally occurring homologous chromosomes in cells, but in somatic cells, the efficiency of gene homologous recombination is particularly low, which increases the workload of gene knockout operations and limits gene knockdown. In addition to the application of technology. Moreover, it is impractical to achieve permanent repair of the human genome with simple homologous recombination, which also seriously hinders the development of biological research.
1.2 Gene knockout using random insertion mutations
Large-scale random insertion mutations theoretically enable knockout of any gene within the genome. This technology can improve the efficiency of gene knockout, make the gene completely inactivated and easy to isolate and identify. At present, more effective gene knockout methods are random insertion mutations which can be divided into T-DNA insertion mutations and transposon insertion mutations, both of which are widely used in plants. T-DNA insertion inactivation technology can produce stable insertion mutations directly in plant genomic DNA, and the insertion site is more random, but only suitable for plants that are easily transformed by T-DNA, and often causes chromosomal weight. The phenotype makes the mutant phenotype unrelated to T-DNA insertion and is difficult to perform genetic analysis. This knockout method produces a 35% to 40% mutation rate in Arabidopsis, and 19% of the mutants have a visually detectable phenotypic characteristic. In recent years, such gene knockout methods have been widely used in Arabidopsis research.
2 Emerging gene knockout technology
2.1 Using gene knockout caused by RNA interference
RNA interference is an RNA-dependent gene silencing phenomenon that is a sequence-specific post-transcriptional gene expression silencing induced by double-stranded RNA molecules at the mRNA level. dsRNA can produce a series of siRNAs with a length of 21-22 nt under the action of Dicer. The siRNA molecule, nuclease and helicase bind to form an RNA-induced silencing complex. R1SC catalyzes the unwinding of double-stranded siRNA in an ATP-dependent manner. The single-stranded siRNA inside RISC is used to recognize the target RNA complementary thereto by base pairing, cleave the target RNA, and degrade by RNase, thereby causing silencing of the target gene. Therefore, gene knockout can also be achieved by introducing a dsRNA molecule into a cell, specifically degrading the mRNA in the cell and homologous thereto, and blocking the expression of the endogenous gene to inactivate the gene.
2.2 Gene knockout of zinc finger nuclease gene targeting technology
The core design idea of ​​zinc finger nuclease gene targeting technology is to fuse two specific functional domains, namely specific recognition modules and functional modules, to form proteins with specific functions. The DNA binding domain of a single ZFN typically contains 3-6 Cys2-His2 zinc finger protein repeat units that specifically recognize one triplet base. The non-specific endonuclease linked to the zinc finger protein group is derived from the DNA cleavage domain of the 96 amino acid residues at the c-terminus of Fokl, and each Fokl monomer is linked to a zinc finger group to form a ZFN, identifying a specific At the site, when the two recognition sites are separated by a distance of 6 to 8 bp, the two monomeric ZFN interactions produce a digestive function. A DNA double-stranded incision is generated at this specific site, and then the incision repair is performed by using the inherent homologous recombination or non-homologous end-ligation repair mechanism of the cell, thereby achieving the purpose of precise point modification. ZFN-mediated gene knockout technology can precisely modify the gene or its surrounding regulatory elements, can construct a good animal model for the study of human diseases, gene knockout rats obtained by pronuclear injection or cytoplasmic injection, gene knockout rabbit. When there is exogenous DNA in the homologous region, homologous recombination repair occurs, and the site-specific knocking of the foreign gene can be achieved.
2.3 Gene knocking caused by TALEN cleavage of specific nucleotide target sequences
TALENs targeted gene knockout technology is a brand new molecular biology tool and is considered a milestone in the development of gene knockout technology. The design and construction of TALENs targeted gene knockout is based on the principle that a transcriptional activator-like effector secreted by the plant pathogen Xanthomium can recognize DNA sequences. Using the sequence module of TAL, it can be assembled into a modular protein that specifically binds to any DNA sequence. The DNA binding domain of the TAL protein is fused to the cleavage domain of Fokl endonuclease, and the target gene is interrupted at a specific site, and DNA manipulation is performed at the site, such as Knock-in, gene knockout. (Knock-nut) or site-directed mutagenesis.
2.4 Cas9 gene knockout technology
In early 2013, a new type of artificial endonuclease clustered rare interspaced short palindromic repeats (CRISPR/Cas9) emerged, mainly from bacteria and archaea through an evolutionary adaptive immune defense system, type II CRISPR/Cas9 The immune system relies on the Cas9 endonuclease family to target and cleave foreign DNA for gene knockout purposes. The Cas9 gene knockout technology is characterized by simple fabrication, low cost and high efficiency. CRISPR/Cas9 will further target gene manipulation to the climax, making multiple gene knockouts and knock-ins easier and more efficient. As a new gene editing technology, CRISPR/Cas9 has the characteristics and advantages of high targeting accuracy, short test period, no species restriction, and good activity. In addition, the CRISPR/Cas9 knockout system can simultaneously perform multiple target simultaneous digestion on multiple sites in the same cell (Es). This technique is used to successfully obtain a model knockout model of zebrafish and other model animals. It is possible to knock out and knock in multiple genes. Although little is known about the specificity and immunogenicity of this technology, it will be greatly improved as the research progresses.
Although the use of artificial nucleases ZFN, TALENs and bacterial adaptive immune system CRISPR is still in the early stages of research, it has shown great potential and broad application prospects in gene knockout, which is considered to be Innovative technologies that target genetic modification. Since these new technologies still have many unknown factors and are not well studied, they will play a limited role in the research of gene knockout techniques.
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