Pineda Michael, Moghadam Farzaneh, Ebrahimkhani Mo R, Kiani Samira
School of Biological and Health Systems Engineering, Arizona State University , Tempe, Arizona 85281, United States.
Center for Regenerative Medicine, Mayo Clinic, Phoenix, Arizona 85054, United States.
ACS Synth Biol. 2017 Sep 15;6(9):1614-1626. doi: 10.1021/acssynbio.7b00011. Epub 2017 Jun 7.
An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.
理想的体内基因治疗平台可提供安全、可重新编程且精确的策略，以高时空分辨率调节细胞和组织的基因调控网络。成簇规律间隔短回文重复序列（CRISPR），一种细菌适应性免疫系统，及其相关蛋白CRISPR相关蛋白9（Cas9），因其能够以前所未有的灵活性和精确性按需靶向和修饰DNA序列而受到关注。Cas9的精确性和可编程性源于其与与所需基因组序列互补的引导RNA（gRNA）的复合。CRISPR系统开启了广泛的应用，包括遗传疾病建模、功能筛选和合成基因调控。使用CRISPR进行体内基因工程的可行性作为下一代体内治疗方法已获得了显著的关注。然而，在基于CRISPR的策略全面实施之前，仍有一些障碍需要克服。一些关键问题集中在CRISPR平台的可控性上，包括最小化基因组脱靶效应、最大化体内基因编辑效率、体内细胞递送以及时空调控。CRISPR系统的可修饰组件：Cas9蛋白、gRNA、递送平台以及所递送的CRISPR系统的形式（DNA、RNA或核糖核蛋白）最近已分别进行了改造，以设计出更好的基因组工程工具箱。本综述着重评估CRISPR作为下一代体内基因治疗平台的潜力，并讨论可应对与这一新兴技术临床转化相关挑战的生物工程进展。
