Cancer Genetics and Epigenetics 2024, Vol.12, No.5, 294-305 http://medscipublisher.com/index.php/cge 295 2 Overview of Gene Editing Technologies 2.1 CRISPR-Cas9 system: mechanism and application Gene editing technologies have transformed biomedical research by allowing precise modifications of the genome, offering immense potential for cancer research and therapy. These technologies can target and modify genes associated with tumor progression and chemoresistance, essential in understanding and treating cancers like ovarian cancer. The main gene-editing tools include CRISPR-Cas9, TALENs, and Zinc Finger Nucleases (ZFNs), each with unique mechanisms and applications. Recently, base editing and prime editing have emerged as more precise alternatives. Collectively, these techniques enable researchers to manipulate cancer-related genes with unparalleled accuracy, advancing both cancer models and therapeutic strategies. Their applications range from creating accurate cancer models to developing targeted therapies, making them crucial in the ongoing battle against cancer. The CRISPR-Cas9 system, derived from a bacterial defense mechanism, is one of the most revolutionary gene editing technologies. It uses a guide RNA to direct the Cas9 enzyme to a specific DNA sequence, creating a double-strand break (DSB). This break can be repaired through non-homologous end joining (NHEJ), which often introduces mutations, or homology-directed repair (HDR), which allows precise gene corrections. Compared to earlier tools like ZFNs and TALENs, CRISPR-Cas9 is easier to design, more efficient, and cost-effective, making it the tool of choice for many genetic studies. In ovarian cancer, CRISPR-Cas9 has been used to knock out key oncogenes and tumor suppressor genes, like TP53 and BRCA1/2, providing new insights into cancer development and progression (Karimian et al., 2019). Additionally, CRISPR-Cas9 facilitates the development of immune therapies such as CAR-T cells and checkpoint inhibitors. However, challenges remain, particularly the risk of off-target effects and the difficulty in delivering the CRISPR-Cas9 system to specific tissues. Advanced techniques like base editing and paired nickases are being developed to minimize these off-target effects and improve the system's safety (Naeem et al., 2020). 2.2 TALENs: precision and use in cancer research Transcription activator-like effector nucleases (TALENs) are gene-editing tools that recognize specific DNA sequences via engineered TAL effector proteins fused to a FokI nuclease. This allows TALENs to introduce site-specific DSBs, triggering cellular repair mechanisms and gene editing. The advantage of TALENs lies in their high specificity, as each TALEN can be designed to target unique sequences with minimal off-target effects. This precision makes TALENs particularly valuable in cancer research, where they are used to model mutations in tumor suppressor genes and oncogenes. In ovarian cancer, TALENs have been used to knock out or correct mutations in genes such as BRCA1 and TP53, helping researchers understand the molecular basis of these mutations and develop targeted therapies (Yamanaka, 2016). While TALENs are more time-consuming and complex to design compared to CRISPR-Cas9, they remain a preferred method when absolute precision is required, especially in therapeutic contexts. Furthermore, TALENs are less prone to off-target effects, making them a reliable tool for gene editing in clinical research. However, due to their complexity and the rise of simpler alternatives like CRISPR-Cas9, TALENs are now less frequently used, though they continue to serve as a critical tool in certain precise genetic applications. 2.3 Zinc finger nucleases (ZFNs): early applications Zinc Finger Nucleases (ZFNs) were the first programmable gene-editing tools developed and have played a foundational role in the evolution of genome editing. ZFNs work by combining the DNA-binding zinc finger domain with the FokI nuclease, which cleaves the DNA. This allows for highly specific gene modifications, but designing ZFNs requires creating a zinc finger domain for each DNA target sequence, a complex and labor-intensive process. ZFNs have been used extensively in cancer research, particularly in the early stages of gene therapy development. In ovarian cancer, ZFNs have been applied to disrupt key oncogenes and repair mutations in tumor suppressor genes, contributing to advances in personalized cancer treatments (Li et al., 2020). Despite their pioneering role, ZFNs have largely been replaced by more user-friendly technologies like CRISPR-Cas9, which is easier to design and implement. However, ZFNs remain in use for applications that
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