As an easy-to-use and powerful gene-editing tool, CRISPR-Cas9 has been widely used in applications ranging from basic research to the treatment of genetic diseases. The development of this technology has made the generation of knockout (KO) cell line models more accessible. Why use knockout cell lines? In this article, we review the various applications of the knockout cell lines, hoping to bring some inspiration for your research.


1. Researching Disease Pathogenesis

When it comes to studying the pathogenesis of diseases, it is crucial to study the gene function of the pathogenic gene. In vitro studies of gene functions often require the use of cell models to investigate the expression regulation of target genes, such as gene overexpression or knockdown expression to respectively achieve the gain of function or loss of function of a target gene. Although gene interference technology is mature for gene loss of function, in many circumstances, RNAi cannot effectively reduce the expression of the target gene. In other cases, the expression is reduced but no changes in cell biological function can be detected. For these reasons, knockout (KO) cells are needed for studying the expression regulation of target genes. Moreover, CRISPR-Cas9 gene-editing technology mediated gene knockout can also target non-coding region gene fragments. In addition to studying the effect of gene KO, we can further study overexpression of the target gene on the established KO cells: by supplementing the normal gene to restore the phenotype, researchers may more fully identify the function of the target gene. Knockout (KO) cell lines can help us gain a better understanding of gene functions or signal pathways.


2. Identify Drug Targets

According to the statistics, less than 10% of the drugs that have entered the clinical research stage are successful in reaching the market, with tumor-related drugs especially low, at under 5%. Lack of effective drug targets is one of the most important reasons for this situation, as finding a proper target can effectively reduce the money and research cycle of drug development. More importantly, finding a right drug target can greatly reduce the risk of failure in drug development. At present, the whole genome gRNA library can be used to screen high-specificity gene targets. For gRNA that disappears or is enriched after treatment administration, it is possible to search for drug sensitivity or drug resistance related gene targets. However, the screening and discovery of drug targets are only the first step of drug development, and a series of effectiveness and safety experiments need to be carried out before in vivo experiments. Therefore, we can establish a targeted gene knockout (KO) cell line, and then observe the phenotype of the cell line after drug administration, to screen or develop drugs that act on the target.


3. Antibody Specificity Verification

The antibody is one of the most commonly used reagents in experiments, which can reveal a variety of changes in biological activities through antigen-antibody interactions. However, the specificity, sensitivity, and functionality of commercial antibodies are not necessarily fully verified. The quality of antibodies also varies among vendors, which leads to inconsistent results. Knockout cell lines can effectively verify the antibody specificity, because specific antibodies (against the gene KO) will not produce signals in the validation results, while wild cell lines may produce specific protein interaction signals. Therefore, knockout cells can be used as a standard for antibody validation to determine the specificity of the antibody.


4. Gene Therapy Research

Gene therapy has been a popular research subject that has experienced rapid development progress in recent years. Currently, there are hundreds of gene editing-mediated clinical trials are being conducted around the world. The common gene therapy methods used include gene modification, gene inactivation, and gene replacement. Example methods include the use of PD-1 gene knockout autologous T cells to treat prostate cancer, esophageal cancer, and renal cell carcinoma; The erythroid-specific enhancer of Bcl11A was knocked out to upregulate the γ Globulin, which can be used as potential treatment for sickle cell disease and β-thalassemia. These studies and clinical trials have all been achieved with the use of CRISPR/Cas9 gene knockout technology.

In short, the knockout (KO) cell line model has a promising application prospects in basic research, drug development, and many other fields. With its advantages – such as complete loss of gene function, regulation of any segment of the genome, and convenient recovery experiments - knockout cell lines are an irreplaceable tool in the research of disease mechanisms and drug targets.


Cyagen’s Knockout Cell Line Services

Cyagen’s Smart-CRISPR™ cell line modeling services enable large fragment knockout  and/or accurate mutation(s) – providing knockout cell lines with complete loss-of-function of the target gene.

Our custom cell line modeling services platform features CRISPR-mediated gene editing that is optimally targeted with our proprietary artificial intelligence (AI)-based AlphaKnockout Smart Gene Targeting System - enabling higher knockout (KO) efficiency. We can perform multiple knockout strategies, including frameshift mutation, large fragment knockout, and multiple genes knockout. With CRISPR/Cas9 technology, researchers can easily solve the problem of residual protein expression seen in RNA interference (RNAi) gene knockdown models.

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