International Journal of Clinical Case Reports 2024, Vol.14, No.2, 94-106 http://medscipublisher.com/index.php/ijccr 95 donor organs, ultimately saving countless lives by providing a reliable alternative through xenotransplantation with genetically modified pigs. 1 Background on CRISPR-Cas9 Technology 1.1 Mechanism and principles of CRISPR-Cas9 CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) is a revolutionary genome-editing tool derived from the adaptive immune system of bacteria and archaea. This system provides these microorganisms with a defense mechanism against invading viruses and plasmids by targeting and cleaving their DNA. The mechanism of CRISPR-Cas9 involves two key components: the Cas9 protein and a guide RNA (gRNA). The gRNA is composed of two parts: a CRISPR RNA (crRNA) that recognizes the target DNA sequence and a trans-activating crRNA (tracrRNA) that binds to the Cas9 protein. Together, they form a complex that scans the genome to find and bind to a specific DNA sequence complementary to the crRNA. Once bound, the Cas9 protein induces a double-strand break at the target site. This break is then repaired by the cell's natural repair mechanisms, either through non-homologous end joining (NHEJ) or homology-directed repair (HDR), allowing for the introduction of specific genetic changes (Wang et al., 2016). 1.2 Advantages of CRISPR-Cas9 over traditional gene editing techniques CRISPR-Cas9 offers several advantages over traditional gene editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). CRISPR-Cas9 requires only a single guide RNA to direct the Cas9 protein to the target DNA, whereas ZFNs and TALENs require the engineering of specific proteins for each target sequence. This makes CRISPR-Cas9 simpler and more cost-effective (Sun et al., 2017). CRISPR-Cas9 can target multiple genes simultaneously by using different gRNAs, allowing for the editing of several genes in a single experiment. This is particularly useful for studies involving complex genetic interactions (Lowder et al., 2015). The CRISPR-Cas9 system can be easily adapted for a variety of applications beyond simple gene editing, including gene activation (CRISPRa), gene repression (CRISPRi), epigenetic modifications, and live-cell imaging of chromosomal loci (Wang and Qi, 2016). High Precision: The ability to design specific guide RNAs for precise targeting of DNA sequences allows CRISPR-Cas9 to introduce genetic modifications with high accuracy, reducing off-target effects compared to other methods (Mollanoori and Teimourian, 2018). 1.3 Applications of CRISPR-Cas9 in biomedical research CRISPR-Cas9 has wide-ranging applications in biomedical research, demonstrating its versatility and transformative impact. CRISPR-Cas9 has been used to create precise genetic models of human diseases in animals. For example, it has facilitated the development of rodent models with specific mutations to study cancer, neurodegenerative diseases, and metabolic disorders (Collins et al., 2017). CRISPR-Cas9 holds promise for therapeutic gene editing to correct genetic defects. Clinical trials are underway to evaluate its efficacy in treating conditions such as sickle cell anemia, beta-thalassemia, and certain types of cancer by editing patient-derived cells (Li et al., 2021). By enabling the systematic disruption of genes, CRISPR-Cas9 has accelerated functional genomics studies, helping to identify genes essential for various biological processes and disease states (Ratan et al., 2018). Beyond medical applications, CRISPR-Cas9 is also revolutionizing agricultural biotechnology by enhancing crop resistance to diseases and environmental stresses, improving yield, and modifying nutritional content (Liu et al., 2017). CRISPR-Cas9 is used in drug discovery and development to identify potential drug targets and understand drug interactions at the genetic level. This helps in the creation of more effective and targeted therapies (He et al., 2022).
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