Both gene knockout and knock-in models are powerful tools widely used in biomedical research to gain important insights into the function of genes and disease mechanisms and support the development of novel therapeutics.
Gene knockout models involve the deliberate inactivation of a specific gene, knock-in models are developed by inserting a specific gene into a particular location within the genome.
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Foundations of Gene Knockout and Knock-in Models
Various methods can be used to knock out or knock-in genes for both in vitro and in vivo models. Ribonucleic acid (RNA) interference (RNAi), for example, is frequently used to silence target genes using small interfering RNA (siRNA) or small hairpin RNA (shRNA), the latter of which is associated with superior efficacy in knocking down genes1.
Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered, regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 systems are common gene-editing technologies, with CRISPR/Cas9 particularly advantageous for these applications.
The two main components of the CRISPR/Cas9 system include guide RNA (gRNA), which identifies and binds to the gene of interest through base pairing, and the Cas9 protein, which induces a DNA double-strand break (DSB)2.
Nuclease-induced DNA DSBs can be repaired through either homology-directed repair (HDR) or nonhomologous end-joining (NHEJ).
Historically, homologous recombination (HR) has been used to add, replace, or inactivate genes; however, this DNA repair approach is limited in its efficiency in both in vivo and in vitro systems. Comparatively, HDR is associated with superior efficiency by using an exogenous DNA template that delivers precise mutations in a site-specific manner3.
Applications in Disease Research
Understanding Disease Mechanisms
Gene editing technologies are powerful tools that allow researchers to examine the function of various genes and manipulate the behavior and functions of specific cells for investigative and therapeutic purposes.
Tools like ZFNs, TALENS, and CRISPR/Cas9 have also facilitated the establishment of genetically engineered animals that resemble various diseases to understand better the molecular mechanisms involved in these conditions and investigate the potential efficacy of novel therapeutic strategies.
Within cancer research, CRISPR/Cas9 has been widely utilized to introduce mutations in tumor suppressor genes and modify oncogenes to generate two- and three-dimensional in vitro models.
Likewise, CRISPR/Cas9 has been pivotal in establishing in vivo cardiovascular disease (CVD) models to investigate novel treatments and ultimately improve current patient care, primarily focused on symptomatic treatment rather than addressing the genetic defects associated with this widespread health condition.
Neurodegenerative diseases, including Huntington’s disease (HD), Alzheimer’s disease (AD), and Parkinson’s disease (PD), can arise due to multiple environmental and genetic factors, either alone or in combination.
The complexity of these diseases warrants sophisticated approaches like genome editing to clarify the genetic interactions that lead to their clinical features.
Infectious disease research has also utilized gene editing technologies to alter host genes a specific virus needs to enter cells or target viral genes involved in viral replication3.
ZFNs, for example, have been used to modify human immunodeficiency virus (HIV) genes to produce CD4+ T-cells that are resistant to the virus and are subsequently reinfused into patients.
Therapeutic Development
Chimeric antigen receptor (CAR) T-cell therapy begins with collecting the patient’s white blood cells. T-cells are isolated and re-engineered with tumour-antigen-specific receptors and co-stimulating molecules3. A CAR-containing viral vector is then transduced into these modified T-cells and infused into the patient.
By inducing the expression of CARs, synthetic receptors, onto these cells, CAR T-cells can effectively recognize and bind to tumor antigens and induce cell death.
Despite over 400 clinical trials that have been conducted on CAR T-cell therapy, this technology is associated with several manufacturing limitations, as well as risks of relapse, toxicity, and drug-resistant issues1.
In addition to cancer, ZFN and CRISPR/Cas9 technologies have been applied in five clinical trials to treat hematological diseases.
In April 2024, the United States Food and Drug Administration (FDA) approved BEQVEZ to treat moderate to severe hemophilia B, a recessive X-linked disease in which coagulation factor IX (F IX) is absent4. BEQVEZ is an adeno-associated virus (AAV)-based gene therapy that introduces transduced cell
Advantages and Limitations
In cancer research, gene knockout and knock-in studies are typically performed on cancer cell lines to investigate the efficacy of an existing drug or facilitate the development of a novel cancer drug. In vitro knockout and knock-in models are also used for phenotypic screens; however, these are expensive and time-consuming experiments5.
Knockout and knock-in in vivo models provide important insights into the normal behavior of a target gene. For example, knockout mice have been crucial for studying and modeling different types of cancer, obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging, and neurodegenerative diseases like Parkinson’s disease.
In addition to allowing researchers to understand the pathophysiology of these diseases, knockout mice also provide important biological insights into the therapeutic efficacy of novel drugs.
Despite their utility, there are various limitations and challenges associated with the reliability of knockout and knock-in mammalian models.
For example, about 15% of gene knockouts are developmentally lethal, thus limiting the ability of researchers to study the behavior of these genes in adult mice6. Furthermore, since certain genes have different functions in mice than in humans, their modification could fail to produce clinically relevant results.
The limitations associated with these pre-clinical models have led researchers to investigate the potential of computational methods like metabolic networks as alternative methods to identify drugs that effectively kill cancer cells5.
In a 2021 Scientific Reports study, researchers utilized genome-scale metabolic models (GSMMs) to predict how the knockout of single metabolic genes would affect the anti-proliferative activity of NCI-60 cells, which led to the identification of 13 potential gene targets for future studies.
Future Directions
Many diseases arise due to single-nucleotide substitutions rather than complete gene disruption. This may prevent current knockout models from accurately replicating the subtle changes in function associated with these genetic mutations.
Thus, precise genome editing (PGE) technologies that can create targeted and specific mutations in endogenous genes are urgently needed.
Although significant advancements have been made in developing in vitro systems transfected with PGE components, translating these technologies into in vivo models remains challenging.
Nevertheless, PGE has been successfully achieved in several model organisms, including mice, zebrafish, rabbits, pigs, and non-human primates7.
References
- Zhang, X., Jin, X., Sun, R., et al. (2022). Gene knockout in cellular immunotherapy: Application and limitaitons. Cancer Letters 540. doi:10.1016/j.canlet.2022.215736.
- Leal, A. F., Herreno-Pachon, A. M., Benincore-Florez, E., et al. (2024). Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches. International Jounral of Molecular Science 25(5); 2456. doi:10.3390/ijms25052456.
- Li, H., Yang, Y., Hong, W., et al. (2020). Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduction and Targeted Therapy 5(1). doi:10.1038/s41392-019-0089-y.
- Dhillon, S. (2024). Fidanacogene Elaparvovec: First Approval. Drugs 84; 479-486. doi:10.1007/s40265-024-02017-4.
- Paul, A., Anand, R., Karmaker, S. P., et al. (2021). Exploring gene knockout strategies to identify potential drug targets using genome-scale metabolic models. Scientific Reports 11(213). doi:10.1038/s41598-020-80561-1.
- Knockout Mice Fact Sheet [Online]. Available from: https://www.genome.gov/about-genomics/fact-sheets/Knockout-Mice-Fact-Sheet.
- Richardson, C., Kelsh, R. N., & Richardson, R. J. (2023). New advances in CRISPR/Cas-mediated precise gene-editing techniques. Disease Models & Mechanisms 16(2). doi:10.1242/dmm.049874.
Further Reading