Gene knockout (KO) cell lines enable researchers to study the role of specific genes by comparing the phenotypic differences among the knockout and wildtype cells. Herein, we have collected the Frequently Asked Questions on knockout cell line generation, hoping to help researchers gain a proper understanding of how knockout cell lines are developed and used.


To achieve gene loss of function, should I perform RNAi knockdown or knockout?

  • When knockout may cause cells to proliferate very slowly or stop growing, or when the detection efficiency of the cell pool stage after cell transfection shows a significant decline, it can be determined that knockout may be fatal, and RNAi is recommended;
  • Due to the existence of some strong functional proteins, even if the expression is downregulated, it still has a strong function. If RNAi cannot produce phenotype all the time, and the previous data shows this gene can produce a phenotype, in this situation, the knockout is recommended. In addition, if the target gene is located in a non-transcriptional region, or the target gene has strong transcriptional efficiency, it cannot be effectively interfered with by RNAi, and knockout is the only choice. Additionally, knockout cells are more suitable for complement experiments.
  • When we use RNAi to perform gene knockdown and observe the change of cell phenotype, further knockouts can still be performed to make the experimental conclusions more convincing.


What is the difference between monoclonal knockout (KO) cell lines and KO cell pools?

In monoclonal (single) knockout (KO) cell clones, all cells are proliferated from one homozygous cell verified by sequencing, and all cells are the same genotype. Knockout cell pool refers to the pool of cells that have not undergone screening and monoclonalization, so it can be understood that this population contains homozygote, heterozygote, and wild-type knockout cells. After obtaining a KO cell pool,  researchers need to screen for single knockout cell clones according to the experimental needs.

When it is necessary to remove interfering factors from a cell population, using a monoclonal stable cell line is recommended due to its relatively pure genomic background yielding consistent experimental results. When performing systems biology studies, cell population factors need to be taken into account. At the same time, many experiments use cell pools (mixed clones) to reduce the interference caused by differences between cells and how the various integration sites of monoclonal cell lines could exhibit inconsistent cell behaviors. Therefore, the options to use monoclonal or mixed clonal cell strains are based on the purpose of the experiment(s) planned.


Frameshift mutation and fragment knockout - which is better for disabling the target protein’s functional domain?

Many people think that fragment knockout has a greater impact on the functional domain of the target protein. In fact, both frameshift mutation and fragment knockout can achieve an ideal knockout effect in many cases.

To determine whether it is a fragment knockout or a frameshift mutation, we should consider whether the knockout fragment is a multiple of non-3. In other words, in addition to the knockout region, we should also consider whether the knockout region can cause a frameshift mutation. If the knockout fragment does not cause a frameshift mutation, the protein only lacks the corresponding amino acid sequence of the knockout region, but other regions of the protein are not affected.


Single gRNA (frameshift)

Double gRNA (fragment)


The design is simple, the plasmid is small, easy to transform, and the cost is low.

It is necessary to design gRNA on exons.

The identification is simple; All gene copies can be deleted uniformly, and the functional analysis is simple.

It is necessary to design gRNA on introns.


Identification is troublesome, sequencing is required, and a non-triple frameshift must be generated, which is difficult to achieve for a few cell lines.

The technology is complex, the cost is high, and the knockout efficiency is low. And more monoclonal analysis is needed to screen homozygous cell lines.


Can we ensure that the protein is not expressed?

From a scientific point of view, it is impossible to ensure that any cell does not express a protein, whether using frameshift mutation or fragment knockout strategies. For example, the frameshift mutant mRNAs may undergo variable splicing and skip the frameshift site for translation, resulting in protein expression. Similarly, the site of the protein’s expressed functional domain is just ahead of the cleavage site – permitting continued expression of functional protein.

Therefore, one cannot hastily guarantee that the protein is not expressed. However, we can avoid the problem of residual protein by optimizing the design of gRNA. By combining optimal gRNA design with antibody analysis and phased WB tests during the experimental process, we can provide you with a complete scientific solution for knockout cell lines, so that you no longer need to worry about the problem of residual protein.


How to design the experimental strategy when performing a genetic rescue experiment

First of all, for knockout cells that need to do a rescue experiment, the strategy of stable transformation of gRNA + frameshift is not recommended. This is because Cas9 continues to express, and gRNA acts on the exon system. When performing genetic rescue experiments, frameshift mutations must be used. The cDNA mutations, namely synonymous mutations, remove the PAM sequence recognized by CRISPR. Otherwise, the cDNA will also be cleaved. In addition, the fragment knockout means that two gRNAs are cleaved on the introns, and the cleavage site is not recognized on the cDNA, so the overexpression recovery experiment is relatively smooth.


What are the tips for accelerating cell line development?

We can increase the serum concentration in the culture medium, or switch to fetal bovine serum, ES serum, or add stimulating factors to the medium; spread a layer of trophoblast cells before plating 96 well for culture.


How can Cyagen 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.

>> Learn More About Cyagen Knockout Cell Line Services