Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia.
General Administration for Infectious Disease Control, Ministry of Health, Riyadh 12382, Saudi Arabia.
Int J Nanomedicine. 2024 Jun 6;19:5335-5363. doi: 10.2147/IJN.S455574. eCollection 2024.
The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.
CRISPR/Cas9 基因组编辑方法是基因工程的一项革命性进展。由于其设计简单、基因组编辑能力强大，为治疗不同的感染性、代谢性和遗传性疾病提供了有前途的策略。Cas9（SpCas9）与 sgRNA 和其靶 DNA 的复合物的 2.5 Å 分辨率晶体结构揭示了一个容纳 sgRNA:DNA 异源双链体的凹槽，其结构为具有靶标识别 (REC) 和核酸酶 (NUC) 结构域的双叶结构。靶标识别、R 环形成和链断裂都需要 PAM 的存在。最近，通过遗传、化学和物理调控策略，极大地改善了 CRISPR/Cas9 基因组编辑的时空控制。使用遗传修饰物抗 Cas9 蛋白、细胞特异性启动子和组蛋白乙酰转移酶，提高了 CRISPR/Cas9 作为下一代基因组编辑工具的应用。此外，通过化学控制、小分子激活剂、寡核苷酸缀合物和生物响应性递送载体的干预，改善了其在生物领域其他领域的应用。此外，通过附着在这种分子工具上的热、光、磁和超声响应元件的物理控制的干预，进一步推动了基因组编辑的发展。这些策略显著降低了 CRISPR/Cas9 的非预期脱靶效应。然而，其他非预期效应仍然为使用这种基因组编辑方法进行全面的临床转化提供了一些挑战。在这篇综述中，我们总结了 CRISPR/Cas9 的结构、机制作用以及小分子激活剂、抑制剂、启动子和物理方法的最新进展。最后，讨论了脱靶测量方法、挑战、未来前景和临床应用。
