International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 193-202 http://medscipublisher.com/index.php/ijmms 195 3 Fundamentals of Gene Editing Technologies Gene editing technologies have revolutionized the field of genetic research and therapy, offering unprecedented precision in modifying the genome. These technologies are particularly promising for treating genetic disorders such as sickle cell anemia (SCA), which is caused by a single point mutation in the beta-globin gene. 3.1 CRISPR-Cas9 technology CRISPR-Cas9 is a groundbreaking gene editing technology that has transformed genetic research and therapeutic applications. The system consists of two key components: the Cas9 enzyme, which acts as molecular scissors to cut DNA, and a guide RNA (gRNA) that directs Cas9 to the specific location in the genome to be edited. This technology allows for precise modifications by creating double-strand breaks (DSBs) at targeted sites, which are then repaired by the cell's natural repair mechanisms, often leading to insertions or deletions (indels) (Ji, 2020; Uchida et al., 2021). CRISPR-Cas9 has shown significant potential in treating sickle cell anemia by targeting the BCL11A gene, which represses fetal hemoglobin (HbF) expression. By disrupting this gene, researchers have successfully increased HbF levels, ameliorating the symptoms of SCA (Zeng et al., 2020; Uchida et al., 2021). However, the technology is not without its challenges, including off-target effects and the risk of unintended mutations (Ji, 2020; Newby et al., 2021). 3.2 Other Gene editing tools In addition to CRISPR-Cas9, several other gene editing tools have been developed, each with unique mechanisms and applications. Transcription Activator-Like Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) are two such technologies that predate CRISPR-Cas9. Both TALENs and ZFNs use engineered proteins to create DSBs at specific genomic locations, which are then repaired by the cell's natural mechanisms (Wilkinson et al., 2021). While TALENs and ZFNs offer high specificity, they are more complex and time-consuming to design compared to CRISPR-Cas9. Nonetheless, they have been successfully used to correct the sickle cell mutation in hematopoietic stem cells, demonstrating their potential in treating genetic disorders (Wilkinson et al., 2021). 3.3 Base and prime editing Base editing and prime editing are recent advancements in the field of gene editing that offer more precise and efficient modifications without creating DSBs. Base editors are fusion proteins that combine a deaminase enzyme with a CRISPR-Cas9 variant, allowing for the direct conversion of one DNA base to another. There are two main types of base editors: cytosine base editors (CBEs) and adenine base editors (ABEs), which enable C-to-T and A-to-G conversions, respectively (Kantor et al., 2020; Chu et al., 2021). Prime editing is a more versatile tool that can introduce all twelve possible base-to-base conversions, as well as small insertions and deletions. Prime editors use a modified Cas9 enzyme fused to a reverse transcriptase, guided by a prime editing guide RNA (pegRNA) to specify the target site and the desired edit (Kantor et al., 2020). This technology has shown promise in correcting the sickle cell mutation with high precision and minimal off-target effects (George et al., 2022). Both base and prime editing hold significant potential for treating sickle cell anemia by directly correcting the point mutation responsible for the disease. These technologies offer a safer and more efficient alternative to traditional gene editing methods, paving the way for new therapeutic strategies (Kantor et al., 2020; George et al., 2022). The advancements in gene editing technologies, particularly CRISPR-Cas9, base editing, and prime editing, have opened new avenues for the treatment of sickle cell anemia. These tools offer precise and efficient methods for correcting genetic mutations, bringing us closer to potential cures for this debilitating disease. 4 Applications of Gene Editing in Sickle Cell Anemia 4.1 Gene editing in hematopoietic stem cells Gene editing in hematopoietic stem cells (HSCs) has emerged as a promising approach to treat sickle cell anemia (SCA). Various gene editing technologies, including CRISPR/Cas9 and base editors, have been employed to
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