There may be confusion about gene knockout and gene interference models because of their similarities. However, gene interference only reduces gene expression at post-transcriptional level and cannot completely remove genes from the genome, as occurs in a gene knockout. Sometimes, if the background is not clean, it may be difficult to analyze the resulting phenotype. If protein expression cannot be decreased in gene interference experim
In the process of mRNA translation into protein, the amino acids that make up the protein are determined by three adjacent bases, known as codons, and the start codon of mRNA is generally AUG. When translation begins, the ribosome starts to translate from start codons, such as AUG and moves, along the mRNA, with every three adjacent bases determining an amino acid.
Fragment knockout refers to the removal of one or more exon regions within a gene to achieve gene disruption. In our platform's fragment knockout workflow, two genomic targeting guides are designed to flank the region intended for deletion. These guides direct the nuclease-mediated cleavage of the genome at the specified sites, enabling removal of the intervening DNA segment.
Some may wonder if it is possible to knockout the whole gene instead of exon(s) only. Technically, it can be achieved, but the knockout of the entire gene fragment is time-consuming and laborious process, and sometimes it can make results unreliable. First, the difficulty of gene knockout will increase with the length of target fragment. Generally, the exon region of gene is relatively short, but the intron region is exceptionally large, which leads to a gene sequence consisting of at least 10 kb or more. In these cases, it becomes very difficult to knock out the whole gene. Moreover, the intron region of a gene may contain the regulatory elements of other genes, which affects the expression of other genes. If the whole gene is knocked out, it could affect the expression of other genes and influence credibility of the experimental results.
In gene knockout studies using cell lines, both frameshift mutation and fragment deletion strategies have their respective advantages depending on the experimental objectives. For many researchers—particularly those new to the field—it is essential to consult a broad range of literature and draw inspiration from existing experimental designs. However, when choosing between frameshift mutations and fragment knockouts, most publications do not explicitly state which method is optimal for achieving gene disruption.
For instance, one study investigating the role of DNA methyltransferases during mammalian development utilized a frameshift mutation approach for gene knockout. Yet, in the methods section, there was no explicit mention of why this strategy was chosen over fragment deletion, nor any comparison between the two.
In another publication focused on metabolic reprogramming in cancer cells, the authors designed two targeting guides to delete a small genomic fragment. Despite this, the study did not specify that a fragment knockout strategy was being employed, leaving room for interpretation.
These examples highlight a broader trend in the literature: researchers often do not emphasize whether frameshift mutations or fragment deletions are preferable. In practice, either method can be effective as long as it reliably disrupts gene function. The choice often depends on technical feasibility, target gene structure, and downstream validation strategies.
We can perform a variety of knockout (KO) strategies to generate a custom cell line model, including frameshift mutation, large fragment knockout, and multiple genes knockout. We will adopt the best knockout strategy to greatly improve the success rate of target gene knockout and the expression efficiency according to each project's unique requirements.
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