Gene editing technology is like a scalpel that can edit and modify target genes to achieve knockout and addition of specific gene fragments. So far, the three generations of ZFN (zinc finger ribonuclease), TALENs (transcription activator-like effector nuclease) and CRISPR/Cas9 have swept the major laboratories at home and abroad? So what are their advantages? What is the comparison between them? Xiao Bian interviewed the director of the Wisten gene editing team with a curious special interview.
Director Shen introduced the principles, advantages, disadvantages and applications of the three generations of gene editing technology to Xiaobian. Xiao Bian is also a biology major, listened to it with gusto, and enjoyed it. I decided to sell it to everyone and briefly introduce the mystery of gene editing technology.
ZFN (zinc finger ribonuclease) acts as a synthetic restriction endonuclease through its zinc finger DNA binding domain and DNA cleavage domain. The researchers engineered DNA binding domains to target different DNA sequences and then specifically cleave them from the DNA cleavage domain. In addition, ZFN technology works in conjunction with intracellular DNA repair mechanisms to allow researchers to edit genomes freely. The advantage of ZFN technology is that it has high specificity and thus avoids immune response, but high specificity brings another drawback. Genetically engineered zinc refers to DNA-binding protein as a problem. Perhaps the reason is Because of the bulky nature of the protein itself and its environmentally dependent properties. In addition, ZNF technology is prone to off-target, leading to cell death or additional mutations.
TALEN edits nucleases for the second generation genome. TAL effector (TALE) can specifically bind to the sequence of interest, and by linking a piece of artificial TALE with FokI nuclease, it constitutes TALEN, a powerful tool with specific genome editing capabilities. Compared with other techniques, TALEN technology largely avoids functional protein off-target because they can bind DNA target sites with high specificity, and the off-target effect is very weak. In addition, compared with ZFN technology, its design and engineering are not so complicated, and it is not as susceptible to the binding of the surrounding connection environment as ZFN. Therefore, TALEN is simpler to use, easier to build, and less expensive. But assembling the TALEN-encoded plasmid is a lengthy, high-intensity process.
The Director of Shen introduced the first two technologies and began to get more and more excited. It turned out to be his team's masterpiece - the CRISPR/Cas9 system. In recent years, the gene editing technology CRISPR/Cas9 has become extremely popular. Since its introduction, it has made up for many shortcomings of traditional gene editing technology, making the “arbitrary editing†of genes easier. Therefore, the CRISPR/Cas9 technology has become the "trump card" of gene editing technology, and it has a momentum to replace it.
The CRISPR-Cas9 technology is inspired by the immune system of bacteria. The CRISPR sequence consists of a number of short and conserved repeats and spacers. A spacer is a variable sequence that corresponds to the sequence of an exogenous genetic material that was previously invaded by the bacterial genome. These spacer sequences are stored in the bacterial genome and are seen as a memory mechanism that triggers the degradation of invading viral DNA by reinfection. It has been several decades since the theory was proposed, but it was not until 2012 that the tools for this technology were available.
The tools of the CRISPR-Cas9 technology are very simple to use, with the fastest design and construction and the lowest cost. Because it does not involve protein engineering, it takes only a few days to build the CRISPR-Cas9 system. However, the CRISPR-Cas9 technology also has some limitations, such as the problem of off-target. Unlike the complex dimer structure of ZFN and TALEN, the CRISPR-Cas9 system has a simpler monomer structure, which can identify homologous sites by base pairing. The specificity is low in the recognition and cleavage of the desired DNA site, and the target mutation verification is very difficult, and it is necessary to scan the whole genome, which is a great workload. The Wisten Bio-CRISPR-Cas9 team successfully knocked out and introduced many genes into bacteria, plants and mammals, and has its own unique insights and rich technical experience.
Xiaobian today has a great harvest for genetic editing. Do you have a deeper understanding of gene editing technology? Which one do you prefer for three generations of technology?
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