A Brief Discussion on Microbial Gene Editing Methods

Gene editing is the process of modifying an organism's genome using gene editing technology to alter its genetic information and phenotypic characteristics. This is achieved efficiently and accurately by inserting, deleting, or replacing specific target genes.

KMD Bioscience offers a variety of gene editing services, including knockout cell lines and point mutant cell lines. These services cover key genes in various fields such as cancer, neurology, and immunity. Each cell line is validated and quality-tested to ensure that all gene-edited cell lines have the correct genotype, are contamination-free and are highly functional.

Common Gene Editing Methods

Gene editing refers to a new technology that makes targeted modifications to the genome. Using this technology, it is possible to pinpoint a specific site in the genome, cut the target DNA segment at this site, and insert a new gene segment. This process not only simulates the natural mutation of the gene but also modifies and edits the original genome, truly achieving "editing genes". Compared with traditional gene targeting technologies based on homologous recombination and embryonic stem cell (ES) technology, new gene editing technologies retain the characteristics of targeted modification, and can be applied to more species with higher efficiency, shorter construction time, and lower cost. Currently, there are three major gene editing technologies, namely zinc-finger nucleases (ZFN) mediated by artificial nucleases, and transcription activator-like efector nucleases. TALEN technology and RNA-guided CRISPR-Cas nuclease technology.

TALENs, ZFNs, and CRISPR/Cas9 technologies are used to repair DNA breaks through non-homologous end joining or homologous directed recombination repair.

BE technology can substitute a specific base pair, enabling the alteration of a base pair at a particular site to another specified base pair.

Cell repair mechanisms integrate the PE into the genome DNA in a single order. Simultaneously, the original sequence is effectively removed through the cell repair mechanism.

Table 1: Comparison of Different Gene-Editing Approaches

The Principles and Applications of Major Gene Editing Tools 

I. Zinc Finger Nucleases (ZFNs)

The DNA recognition domain composed of ZFP can recognize and bind to specific sites, while the cutting domain composed of FokI can perform the cutting function. The combination of the two can cause double-strand breaks (DSBs) at target sites. Thus, cells can repair DNA through the homologous recombination (HR) repair mechanism and the non-homologous end joining (NHEJ) repair mechanism. HR repair may carry out restoration modification or insertion modification on the target site, while NHEJ repair is prone to insertion mutation or deletion mutation. Both can cause frameshift mutations and thus achieve the purpose of gene knockout. Because FokI endonuclease needs to form a dimer to be active, the chance of missing the target generated by random cutting will be greatly reduced. Users only need to design 8-10 zinc finger domains for target genes, and then combine these zinc finger domains with endonuclease to form ZFNS targeting specific sites. After these ZFNs are delivered to the nucleus of the parent cell line by transfection, electroporation, or viral transport, the two complementary ZFNs will recognize 5' to 3' and 3' to 5' DNA in a tail-to-tail fashion via the zinc finger structure at the target site. Subsequently, the two FokI nucleases form a dimer, activate endonuclease activity to cut target sites, form DNA DSBs, and thus induce the DNA damage repair mechanism.

II. Transcription Activator-Like Effector Nucleases (TALEN)

TALEN is a gene editing tool consisting of transcription activator-like efector (TALE) instead of ZF as the DNA-binding domain and the cleavage domain of FokI nuclease. TALE is derived from the plant Xanthomonas and consists of more than 12 DNA tandem repeat units with recognition specificity and N-terminal and C-terminal sequences on both sides. Each repeating unit generally contains 34 amino acid residues, of which the 12th and 13th amino acids are highly variable and are called repetition-variable diresidue (RVD). Then, the two adjacent target recognition modules were fused to the N-terminal of FokI to form a eukaryotic expression vector, and a TALEN plasmid pair was obtained.

When the TALEN plasmid pair is transformed into cells, the expressed fusion protein will bind to the target site separately and then be cut by the dimerized FokI to complete the gene editing operation. The repetitive amino acid sequence module composed of TAIE can bind specifically to a single base, so it is theoretically possible to design and recognize any target DNA sequence.

III. CRISPR/Cas System-Mediated Gene Editing

The CRISPR-Cas9 system causes double-strand breaks at target sites by recognizing target sequences and cutting nucleases. Therefore, in theory, by replacing the DNA functional recognition domain and endonuclease with sRNA and Cas9, any genome can be edited.

Common Questions and Considerations for Gene Editing

Gene knockout is an indispensable logical step to verify gene function in live animals, but the traditional gene knockout method needs to go through a series of steps such as complex target vector construction, ES cell screening, and chimeric animal model breeding, and its success rate is limited by many factors. Even in mature laboratories, it usually takes more than a year to knock out a gene in a rat or mouse using traditional techniques. The new gene-editing technology is not, knocking out genes is efficient and fast, and is a powerful tool for studying gene function.

I. How can off-target effects be prevented?

Develop and utilize effective tools that can accurately predict off-target effects and create new gene editing systems. Enhance methods for delivering gene-editing tools by improving system efficiency. By designing precise targets based on high-quality reference genomes, we can effectively reduce or avoid the occurrence of missed targets.

II. In terms of damaging the function of the target protein structure domain, which way is more effective: frameshift mutations or fragment knockout?

When deleting a fragment, it is necessary to consider not only the deleted region itself but also whether the deletion of the region will cause coding mutations. If the deleted fragment fails to cause a coding mutation, the protein simply lacks the amino acid sequence corresponding to the deleted region, and its function may not be affected.

III. Why is there low efficiency in obtaining point mutation/KI- positive cell clones?

KI cell line construction is influenced by various factors, such as the type and length of the gene, homologous recombination efficiency, and cell types, which can affect the efficiency of editors. These factors also contribute to the high technical difficulties associated with KI.

We designed a CRISPR-Cas9 system or TALEN construct with homologous recombination, random integration, site-specific integration, precise cutting, knock in, and labeling function, and were able to efficiently transfect it into cells and validate genotype and phenotypic outcomes. By using our homologous recombination, random integration, and site-specific integration products, solutions, and tools, you can avoid trial and error and get results quickly and easily.

References

[1] Xu, R., Liu, X., Li, J., Qin, R., and Wei, P. (2021). Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice. Nat Plants 7, 888-892.

[2] Lin, Q., Jin, S., Zong, Y., Yu, H., Zhu, Z., Liu, G., Kou, L., Wang, Y., Qiu, J.L., Li, J., and Gao, C. (2023). High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol 39, 923-927.

[3] Xu, W., Yang, Y., Yang, B., Krueger, C.J., Xiao, Q., Zhao, S., Zhang, L., Kang, G., Wang, F., Yi, H., Ren, W., Li, L., He, X., Zhang, C., Zhang, B., Zhao, J., and Yang, J. (2022). A design optimized prime editor with expanded scope and capability in plants. Nat Plants 8, 45-52.