The traditional approaches used in gene function studies, especially in cell lines, include gene interference, gene overexpression, and gene knockout. Herein, we will focus on discussing the most popular approach, gene knockout – a mutation that inactivates a gene function.

Comparing Gene Knockout and Gene Interference Models

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 experiments, and a phenotypic difference cannot be found – a gene knockout approach would be used.

What is a Frameshift Mutation?

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.

When a frameshift mutation occurs, the number of bases in the genome increases or decreases in a non-3 multiple, which shifts the codon reading frame. In this case, protein translation still starts from the start codon of AUG, but due to the addition or deletion of bases, the original gene sequence is destroyed, resulting in a complete change of the expressed protein sequence and even causing protein translation to end prematurely. In either case, the protein expressed by the original gene no longer exists, so we can consider that the gene has been knocked out.

What about situations wherein the base number to be added or deleted is a multiple of 3? This situation cannot be regarded as the frameshift mutation, unless the added or deleted base sequence is in the key functional domain of the protein. Otherwise, the protein obtained afterwards is still functional, so it cannot be regarded as a successful gene knockout. Theoretically, the incidence of non-frameshift mutation is 1/3, but in the process of gene knockout, we can screen out the mutant sequence to be a multiple of non-3 by sequencing, so as to ensure that the positive cloned cells have a frameshift mutation which results in a gene knockout.

What is Fragment Knockout?

Fragment knockout is defined by the deletion of one or some exon regions of a gene through gene knockout. In the process of using CRISRP-Pro to knock out fragments, we will design two sgRNAs at both ends of the fragment to be knocked out, and then cut the genome with the Cas9 protein, so as to knockout the DNA fragment(s).

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.

Which is the Optimal Choice, Frameshift Mutation or Fragment Knockout?

In cell line gene knockout strategies, frameshift mutation and fragment knockout may provide unique advantages or disadvantages depending on the research application. For most beginning researchers, it is a necessary to read a lot of literature and refer to other researcher's experimental ideas. In the selection of frameshift mutation and fragment knockout, the published articles rarely clearly indicate which strategy is optimal choice in gene knockout. For example, in the below article on the function of methyltransferase DNMT3, the author used frameshift mutation approach. However, in the section of experimental methods, there is no clear indication whether to use frameshift mutation or fragment knockout.

In the following article, the author designed two sgRNAs to knock out small fragments of the gene at the same time but did not clearly indicate that the fragment knockout strategy was used.

From this, we can see that many researchers are not concerned with the problem of which is better, frameshift mutation or fragment knockout. Frameshift mutations and fragment knockout are both good strategies as long as they can achieve gene knockout.

How Cyagen Can Help You with Gene Knockout Cell Lines?

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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