Genome editing is the effective design and efficient transformation of the cells in the genome scale, such as multiple sites on genome are simultaneously inserted or deleted so as to optimize the multiple metabolic branch pathways. The genome-scaled efficient editing techniques mainly include synchronous rewriting of multiple sites on the genome, efficient insert, replace or delete, fragment pasting and self-editing.
ZFNs genome editing technology
ZFNs mediated genome editing mainly depends on the zinc finger domain (ZF) recognizing specific sequence of DNA and endonuclease Fok I with low specificity. ZFNs recognizes and cleaves specific sites on the genome to form double strand breaks, greatly increasing the recombination repair frequency of the sites, including non-homologous end links and homologous recombination. Effectiveness of this method depends on the construction of zinc finger domain with specific recognition site of DNA. The structure consists of a plurality of zinc finger domains, and each zinc finger domain contains about 30 amino acids to identify 3 pairs of base sequence. Different zinc finger assembly can identify different sequences. However, zinc finger domain can not be randomly assembled. It must use various design methods and optimization, which is time-consuming; many engineered zinc finger domain is not high in specificity, which is prone to miss cutting. Therefore, the random edit of ZFNs in whole genome has certain limitations.
Genome editing technology mediated by TALENs
TALENs genome editing efficiency is closely related to the efficiency of the assembly that the DNA recognizes domain repeat unit. There are a number of research groups constructing some common carriers, online design software and high-throughput automated bead assembly to improve the efficiency of TALENs. Apart from that it has been widely used in multicellular eukaryotes, this technology has also been applied to yeast genome efficiently and precisely.
Genome editing technology mediated by CRISPR-Cas
Compared with the TALE nuclease, CRISPR-Cas system is more simple and convenient, and easy to operate and expand. CRISPR-Cas system is a prokaryotic immune system that many bacteria and archaea have to defense exogenous DNA (phage and plasmid) invasion. Because the system is easy to operate and it is transformed into genome editing tool.
The application of these technologies can efficiently build excellent performance of the production of bacteria, promote the traditional fermentation industry innovation, and promote new energy and new biomaterials based on the development of new industrial biotechnology.
According to high throughput synthesis technology of DNA and efficient genetic manipulation tools, genome editing technology can more effectively solve many bottleneck problems in the transformation of industrial microorganisms, including genome combination optimization of multiple sites, large fragment of genome modification and complex phenotype transformation. The new application of genome editing technology greatly enhances the speed of the industrial transformation, becoming a hot research field of application.
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