Methods to Facilitate the Development of Oncolytic Bacteria Genome Editing

Posted October 13, 2021 by beauty33

Microbial genome editing is a site-specific chromosome modification. It is one of the most useful techniques in research and plays an important role in metabolic engineering and molecular biology research.
Microbial genome editing is a site-specific chromosome modification. It is one of the most useful techniques in research and plays an important role in metabolic engineering and molecular biology research. Current methods include gene targeting based on homologous recombination, zinc finger nuclease (ZFN) and transcriptional activation-like effector nuclease (TALEN). However, the use of these technologies is often inefficient, time-consuming, laborious, or expensive. Therefore, there is still a need for a fast and simple method that enables precise genome editing at the lowest cost.

The CRISPR/Cas9 system has been widely used in eukaryotic genomes, but its application in bacterial genomes has been seldom studied. Researchers first used the CRISPR/Cas9 system to edit bacterial genomes. This work is a huge breakthrough in microbial genome editing, but high editing efficiency has not been achieved in E. coli. Recently, researchers have achieved very good results in the development of Cas9-based genome editing technology. Although these methods have improved genome editing technology, they can be further improved in certain areas, such as editing efficiency, laboratory time, ease of use, and standard protocols.

In order to solve these problems, researchers began to develop a simple, fast, and efficient method for editing bacterial genomes. For this reason, the multi-plasmid system was tested and found to be relatively laborious and time-consuming. Low editing efficiency has been observed using the plasmid/linear DNA system, especially for wild-type and E. coli cell factory strains. This may be due to their powerful restriction system that degrades linear donor DNA before integrating into the chromosome. In order to avoid these shortcomings, the researchers' strategy is designed to use only one plasmid to edit the genome, which carries all the functional parts, making the technology very easy and fast to practice and achieve high efficiency.

Compared with other CRISPR-based genome modification systems, the single-plasmid system has the advantages of quick operation and high efficiency. Due to the simplicity of a plasmid, there is no need for any other supplementary DNA materials. The experimental process is simpler than other methods, less time-consuming, and can be completed in only 3 days. In addition, the labor intensity is relatively low, and the main tasks include only one plasmid construction and one transformation, as well as the editing of plasmids and the colony screening of editing strains. The researchers conducted a thorough experiment and chose an optimized plasmid assembly strategy, which includes ready-made parts and a fixed linker for editing the plasmid construction. Due to this modular design of plasmid construction, plasmid construction becomes very easy and economical. The preferred editing protocol uses short homology arms to achieve the simplest plasmid construction, but at the expense of some editing efficiency. However, at least one successfully edited strain can be obtained from seven to eight colonies. In addition, colony PCR screening is not laborious and can be performed even in experiments with high editing efficiency.

This method also has the advantage of editing multiple loci. The system only requires a temperature-sensitive plasmid, which makes the process of plasmid solidification simple and quick. In the continuous process of using this technology, the X locus can be edited in 3× days. This quick and simple method of editing the E. coli genome has been used in some studies.

High-throughput and automated genome editing is a technology that molecular biologists need very much. Due to its modular design, this method can be used to develop automated genome editing technologies. In the plasmid construction process, DNA annealing and Golden Gate assembly processes can be easily transformed into computer-controlled practices through the liquid handling platform. The plasmid transformation and induction process can be automated as a MAGE system. The colony PCR screening process can also be carried out by an automated system.

In the research, the goal of developing a very fast, simple and efficient genome editing technology based on the CRISPR/Cas9 system was achieved. This technology only requires one plasmid construction and one transformation work, and can be performed continuously on multiple sites.
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Country United States
Categories Biotech
Last Updated October 13, 2021