A Brief Introduction to Microbial Gene Editing

Microbial gene editing involves modifying the genetic material of microorganisms to achieve specific experimental goals. By manipulating the microbial genome, we can enhance its metabolic pathways to improve the efficiency of synthesizing beneficial compounds like drugs, biofuels, and other valuable substances. Furthermore, this technique can potentially expedite environmental pollutants' degradation by enhancing microbial capabilities to break them down.

KMD Bioscience offers a variety of gene editing solutions, including knockout and point mutant cell lines. These services are tailored to different fields such as cancer, essential genes in the nervous and immune systems, and other related areas. Each cell line undergoes thorough validation and quality assessment to guarantee precise genotypes, absence of contamination, and maximum efficiency.

Classification of Gene Editing

At present, three types of gene expression regulation methods have been developed, including gene silencing, gene overexpression, and gene editing.

Gene overexpression or gene silencing refers to the artificial increase or decrease of the overall expression level of a gene. This is done to observe the changes in biological functions resulting from gene up-regulation or down-regulation and to study the related biological functions of the gene.

A gene expression, or gene overexpression, CDS is used in gene cloning to insert the corresponding gene into a plasmid or viral vector. The carrier frame is constructed with regulatory elements, allowing the gene to be transcribed and translated in large quantities under artificial control, thereby achieving the desired gene expression.

We know that gene editing technology is primarily utilized in agriculture and disease treatment. In agriculture, it is mainly used for crop breeding and improving livestock and poultry breeds. In the field of disease treatment, it is primarily employed in researching and developing related medicines to treat patients.

The goal of gene editing is to change specific gene sequences at the genome level to study the effects of these sequences on biological functions.

The difference between various microbial gene editing techniques 

Bacterial Gene Editing: The CRISPR system was first discovered in bacteria. It originated from the natural immune mechanism of bacteria against viral invasion, allowing bacteria to recognize and eliminate foreign DNA.

Fungal Gene Editing: Fungal genetic transformation can be achieved by introducing an exogenous plasmid into specific gene receptor strains, enhancing the strain's unique abilities. Current methods of import mainly involve fungal DNA transposons, CaCl2-mediated fungal genetic variations, polyethylene glycol (PEG)-mediated gene editing, gene targeting, electric excitation reforming process, agrobacterium-mediated process, and restriction enzyme-mediated methods.

Microbial Transformation of Samples

I. Vaccination involves placing microbes in a suitable artificial medium for breeding or a process known as inoculation.

After high-pressure sterilization processing of the culture medium, tools used for sterilization in an aseptic environment (such as vaccination needles and straws) will contain microbial materials (such as samples, bacterial moss, or bacterial suspension) for vaccination into the culture medium. This process is referred to as sterile operation.

II. Purification of mixed cultures refers to containing a variety of microbial cultures. If all cells in a colony come from the same parental cells, then the colonies can be called a pure culture.

III. Training: According to different oxygen demands, training can be categorized into two types: anaerobic and anaerobic-aerobic. Based on the physical properties of the culture medium, it can be classified into two main categories: solid cultivation and liquid.

IV. Translate: Refers to the plasmid DNA and chromosome-free DNA molecules being transferred into other cells to achieve gene transfer. The recipient cells can naturally contain DNA or can be made sensitive to DNA after human intervention.

Figure 1. The flow chart of microbial gene editing

Validation of Gene Editing

I. Validation Method

After the expression level of a gene is increased or decreased in cells, tissues, and organisms, common verification techniques include gene knockout of DNA (such as CRISPR and its related derivative technology), interference of RNA (RNAi), and regulation of protein levels through Trim-Away and other methods.

II. Content to Deliver

Gene editing vector plasmid and an E. coli culture；

Analysis of sequencing results；

Lab report detailing the experimental steps and bioinformatics analysis results of the gene editing strain；

Provide the corresponding number of gene editing strains and control strains.

KMD Bioscience has successfully developed a variety of technical methods, such as a novel gene sequence synthesis method and the development and utilization of fluorescence clone selection carriers. Through collaborations with prestigious foreign universities, we are establishing an autonomous platform for automated gene sequence analysis, optimization, and examination. These endeavors have equipped us with globally advanced technology distinguished by its distinctiveness.

Our technical team members have diligently mastered each step of the experiment to ensure the precision of the target sequence. Furthermore, we possess the capability to introduce single or multiple mutations at any desired position, in conjunction with gene synthesis, providing flexibility for tailored approaches that optimize cost-effectiveness. Concurrently, our services encompass a comprehensive array of upstream and downstream procedures such as gene sequencing, subcloning, flexible vector selection, and plasmid preparation.

References：

[1] Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2024;533(7603):420-424.

[2] Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2022;353(6305): aaf8729.

[3] Gaudelli NM, Komor AC, Rees HA, et al. Publisher Correction: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2023;559(7714): E8.