{"id":1811,"date":"2023-07-20T20:20:06","date_gmt":"2023-07-20T20:20:06","guid":{"rendered":"https:\/\/www.idtdna.com\/page\/customizing-car-t-cells-using-the-crispr-cas9-system"},"modified":"2025-08-22T16:22:54","modified_gmt":"2025-08-22T16:22:54","slug":"customizing-car-t-cells-using-the-crispr-cas9-system","status":"publish","type":"post","link":"https:\/\/test.idtdna.com\/page\/support-and-education\/decoded-plus\/customizing-car-t-cells-using-the-crispr-cas9-system\/","title":{"rendered":"Customizing CAR-T cells using the CRISPR-Cas9 system"},"content":{"rendered":"

Cancer is a leading cause of death worldwide with more than 10 million deaths per year [1<\/a>]. Despite significant advances in cancer research, current modalities such as chemotherapy, radiation, and surgery have limitations due to their nonspecific targeting and significant side effects. A relatively new approach to combat cancer, called immunotherapy, aims to stimulate the immune system to recognize and destroy only cancer cells, leaving healthy cells intact. One of the most promising approaches to immunotherapy involves the use of T cells that have been genetically engineered to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens.<\/p>\n

The CRISPR\u2011Cas9 system is a precise and efficient gene\u2011editing tool that is being utilized to develop CAR\u2011T cells with enhanced antitumor activity. CRISPR editing enables the specific customization of T cells to enhance their functionality and improve their ability to recognize and destroy cancer cells.<\/p>\n

What are CAR-T cells?<\/h2>\n

Normal T cells mediate key parts of the adaptive cell-mediated immune response. Before they mature and release from the thymus, T cell receptors must go through a checkpoint screening. During this process, the thymocytes which recognize “self” antigens are removed by apoptosis while those that recognize “non-self” antigens mature and move to the spleen or lymph nodes—where they are exposed to foreign antigens via the major histocompatibility complex (MHC) of antigen-presenting cells. In addition, activated T cells release cytokines such as IFN\u2011gamma, TNF\u2011alpha, and TNF\u2011beta.<\/p>\n

In contrast to normal T cells, chimeric antigen receptor (CAR) T cells are engineered via either viral delivery or non-viral methods to express a recombinant gene for CAR, consisting of three domains: (1) extracellular domain with the antigen recognition region; (2) transmembrane domain; and (3) the intracellular domain that has three immunoreceptor tyrosine-based activation motifs (ITAMs), costimulatory molecules (CM1) such as CD28, and an interleukin-12 (IL-12) domain that stimulates the innate immune system (Figure 1). <\/p>\n

\"Recombinant
Figure 1. Recombinant CARs contain four domains. A CAR has an extracellular antigen recognition site, hinge, transmembrane domain, and an intracellular domain. The extracellular domain has a single-chain variable domain that recognizes the tumor cell. Although the intracellular domains have evolved, the fourth generation of receptors contains a costimulatory molecule (CM1) domain such as CD28 or 4-1BB that improve proliferation and sustained response. The interleukin-12 (IL-12) domain attracts and activates innate immune cells. Finally, the three immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated by a tyrosine kinase upon antigen binding, thus activating the intracellular signaling pathways associated with T cell attack on the cancer cell.<\/figcaption><\/figure>\n<\/p>\n

How does CRISPR-Cas9 gene editing create the desired CAR-T cells?<\/h2>\n

The CRISPR\u2011Cas9 system is an efficient gene-editing technique that can be used for “knocking in” or “knocking out” genes. Researchers can use CRISPR to insert CAR genes in a precise location of the T\u2011cell genome. T\u2011cell receptor alpha chain<\/em> (TRAC)<\/em> is a region of the genome consisting of the endogenous T\u2011cell receptor gene. The CRISPR\u2011Cas9 system can efficiently edit the existing T\u2011cell receptor gene through the insertion of the tumor-targeting CAR gene. Experiments have demonstrated that the insertion of CAR genes in the TRAC<\/em> region using the CRISPR-Cas9 system shows high CAR expression and improved anti-tumor activity [2<\/a>].<\/p>\n

Researchers would like to find ways for the CAR\u2011T cells to be standardized, a so called “off-the-shelf” allogenic source. Developing new technology is necessary to eliminate potential immune responses which can contribute to the risk of rejection. Universal allogeneic T cells have been created using CRISPR\u2011Cas9 gene editing to target programmed cell death 1<\/em> (PD-1<\/em>), T cell receptor alpha constant (TRAC), <\/em>and beta\u20112\u2011microglobulin<\/em> (β2M<\/em>). Targeting PD\u20111<\/em> and TRAC <\/em>has been shown to improve T cell effector activities which abrogates tumor growth and improves CAR\u2011T cell efficacy [3,4<\/a>]. Moreover, researchers determined that targeting β2M, a key subunit of the human leukocyte antigen class\u20111 (HLA\u20111) protein, allows for a higher persistence of CAR\u2011T cells in vivo<\/em> [3<\/a>].<\/p>\n

What are the advantages of CRISPR\u2011Cas9 and CAR\u2011T compared to other immunotherapies such as immune checkpoint inhibitors?<\/h2>\n

FDA\u2011approved cancer immunotherapies include adoptive T cell therapy (ACT) and immune checkpoint inhibitors (ICIs) that target cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death ligand (PD\u2011L1), and PD-1. The activation of these receptors can initiate an immune response leading to adverse effects [5<\/a>].<\/p>\n

The CRISPR\u2011Cas9 gene editing technique has been demonstrated to generate CAR\u2011T cells with precise antigen recognition, minimal toxicity, anti\u2011tumor effects, stimulate cytokine production, and inhibit checkpoint molecules [6,7<\/a>].<\/p>\n

What are the remaining challenges of using CRISPR\u2011Cas9 technique for generating CAR\u2011T cells?<\/h2>\n

Although the development of CAR-T cells using universal allogeneic T cells is promising, there are still several challenges that need to be addressed. This includes developing a simplified protocol for delivering sgRNAs and Cas9 while maintaining cell survival after gene manipulation. Also, the potential risks of using retroviruses, lentiviruses, adenovirus\u2011associated viruses (AAV) as viral vectors for delivering CRISPR\u2011Cas9 components to cells. Other challenges of CRISPR\u2011Cas9\u2011mediated editing of CAR\u2011T cells are off\u2011target effects and toxicity. Unintended editing could occur at locations other than the target sites in the genome. These off\u2011target effects can lead to unintended changes in gene expression, translocations, and gross chromosomal aberrations which potentially causes toxicity and other adverse effects [8<\/a>]. Therefore, it is essential to carefully design and validate CRISPR\u2011Cas9\u2011mediated edits to minimize the risk of off-target effects in CAR\u2011T cells.<\/p>\n

Conclusion<\/h2>\n

CRISPR-Cas9 gene editing is a more effective approach for manufacturing CAR\u2011T cells using universal “off-the-shelf” allogeneic T cells. Performing CRISPR\u2011Cas9 for generating CAR\u2011T cells is promising; however, challenges do exist when considering this approach.<\/p>\n

Download our CRISPR Therapeutics eBook<\/h2>\n

CRISPR Therapeutics (PDF) is designed to explore the vast possibilities of Genomic Medicine. It will help translational research and cell and gene therapy developers discover what we have to offer with our cGMP\/q7 capabilities for developing CRISPR-Cas9 gene therapies.<\/p>\n

Download <\/strong>your copy of CRISPR Therapeutics eBook<\/a>.<\/p>\n

\"25-GE_DECODED-BANNER-CRISPR-Therapeutics-eBook-15399\"<\/p>\n","protected":false},"excerpt":{"rendered":"

Cancer is a leading cause of death worldwide with more than 10 million deaths per year [1]. Despite significant advances in cancer research, current modalities such as chemotherapy, radiation, and surgery have limitations due to their nonspecific targeting and significant side effects. A relatively new approach to combat cancer, called immunotherapy, aims to stimulate the […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"ct_builder_shortcodes":"","ct_template_type":"","ct_parent_template":0,"inline_featured_image":false,"footnotes":""},"class_list":["post-1811","post","type-post","status-publish","format-standard","hentry"],"acf":[],"yoast_head":"\nCustomizing CAR-T cell-based applications using the CRISPR-Cas9 system | IDT<\/title>\n<meta name=\"description\" content=\"Learn more about how to use CRISPR-Cas9 system for CAR-T Cell based applications\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/test.idtdna.com\/page\/support-and-education\/decoded-plus\/customizing-car-t-cells-using-the-crispr-cas9-system\" \/>\n<meta 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