通过聚集规则间隔短回文重复(CRISPR)/相关蛋白9(CRISPR/Cas9)的基因组编辑方法是基因工程中的革命性进步。由于其简单的设计和强大的基因组编辑能力,它为治疗不同的传染病提供了一个有前途的策略,新陈代谢,和遗传疾病。化脓性链球菌Cas9(SpCas9)的晶体结构与sgRNA及其目标DNA的复合分辨率为2.5µ,揭示了在具有目标识别(REC)和核酸酶(NUC)结构域的双叶酸盐结构内的沟槽容纳sgRNA:DNA异源双链体。目标识别需要PAM的存在,R环形成,断线。最近,通过遗传,CRISPR/Cas9基因组编辑的时空控制得到了相当大的改善,化学,和物理监管策略。使用遗传修饰抗CRISPR蛋白,细胞特异性启动子,和组蛋白乙酰转移酶提升了CRISPR/Cas9作为下一代基因组编辑工具的应用。此外,化学控制干预,小分子活化剂,寡核苷酸缀合物和生物响应递送载体改善了其在生物领域的其他领域的应用。此外,通过使用热量进行物理控制的中介,light-,磁性-,与这种分子工具相连的超声响应元件进一步彻底改变了基因组编辑。这些策略显著降低了CRISPR/Cas9的不良脱靶效应。然而,其他不良效应仍然为使用这种基因组编辑方法进行全面的临床翻译提供了一些挑战.在这次审查中,我们总结了CRISPR/Cas9结构的最新进展,机械行动,以及小分子活化剂的作用,抑制剂,promotors,和物理方法。最后,脱靶测量方法,挑战,未来的前景,并对临床应用进行了讨论。
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 Streptococcus pyogenes 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.