![]() 23 Therefore, researchers searched for possible ways to modify Cas9 by elucidating the physicochemical structure of Cas9, the mechanism of action by which Cas9 cleaves double chains, and other properties. 20, 21, 22 The base sequence of the original gene is damaged, resulting in inactivation, but the inactivation of a single deleterious gene cannot address the complex processes of all disease events. ![]() Wild-type Cas9 only cuts double-stranded DNA to form double-strand breaks (DSBs), which are repaired through DNA repair mechanisms, namely, homology-directed repair (HDR) and nonhomologous end joining (NHEJ). Promising applications for this technology include the treatment of cancers, cardiovascular diseases, sickle cell anemia, and neurodegenerative disease. 5, 15 However, some highly chromatinized regions in the genome may not be accessible to CRISPR/Cas9. 10, 11, 12, 13, 14 By changing the nucleotide sequence of a small segment of guide RNA, CRISPR/Cas9 allows the accurate targeting of almost any desired genomic locus for the purpose of correcting disease-causing mutations or silencing genes associated with disease onset. 9 The preferred PAM by Streptococcus pyogenes Cas9 (SpCas9) is NGG, which is common in the genomes of most organisms, thereby facilitating the use of CRISPR technology across the fields of plant and animal science, together with biomedicine. The recognition process of the sgRNA requires the involvement of protospacer-adjacent motifs (PAMs), a short guanine-enriched sequence. After a series of processing and maturation processes to generate a single guide RNA (sgRNA), the sgRNA guides Cas9 to shear the DNA strand that disrupts the homologous spacer region. If reinfected with homologous DNA, the bacterium will initiate transcription of the CRISPR region. 7, 8 Bacteria or archaea acquire a segment of their DNA sequence to insert into the CRISPR spacer region when first infiltrated by an exogenous phage or plasmid. 6 The CRISPR/Cas9 system evolved naturally in bacteria and archaea as a defense mechanism against phage infection and plasmid transfer. 5ĬRISPR/ Cas9 is a highly effective gene-editing tool that is widely used in the scientific community. 4 Unlike ZFNs and TALENs, which use proteins to target DNA strands, CRISPR technology directs Cas proteins to a specified location in the genome by changing the base sequence of a small segment of guide RNA, thus improving the efficiency of gene editing and expanding the applicability of gene-editing technology. ![]() 3 To date, gene-editing technology has undergone three main generations of development: the first generation of gene-editing technology was zinc-finger nucleases (ZFNs) the second generation was transcription activator-like effector nucleases (TALENs) and the most widely used third generation gene-editing technology is clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas). ![]() 1, 2 Many diseases are accompanied by changes in gene expression in vivo, particularly some genetic diseases caused by mutations in a single gene, and gene-editing technology is expected to control the occurrence of diseases at the genetic level. Gene editing is a technology that precisely modifies the genome sequence to induce insertions, deletions, or base substitutions in the genome. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency.
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