Chemical Biology and Therapeutics Science , Broad Institute of MIT and Harvard , Cambridge , Massachusetts 02142 , United States.
Department of Medicine , Harvard Medical School , Boston , Massachusetts 02115 , United States.
Biochemistry. 2019 Jan 29;58(4):234-244. doi: 10.1021/acs.biochem.8b01202. Epub 2019 Jan 22.
The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.
CRISPR（成簇规律间隔短回文重复）-Cas 系统是细菌的一种适应性免疫系统，它提供了几种 RNA 指导的 DNA 内切酶（例如 Cas9），这些内切酶正在彻底改变基因组工程领域。Cas9 正被用于进行基因组改变以及基因驱动，其中特定的特征可以通过靶向物种群体在数代中传播。将催化失活的 Cas9 靶向任何基因组位点的简便性导致了碱基编辑、染色质成像和建模、表观遗传编辑和基因调控等技术的发展。毫不奇怪，Cas9 正在生物技术和生物医学研究的众多应用中得到开发，并作为多种病理的基因治疗剂。在这些应用中，需要对 Cas9 的活性进行精确控制，包括剂量、时间和空间等多个维度。这些精确控制对于 Cas9 来说非常重要，因为脱靶效应、染色体易位、免疫原性反应、遗传毒性和胚胎嵌合现象在 Cas9 活性升高和延长时会以更高的水平出现。在这里，我们提供了一种观点，即使用外部刺激（例如小分子或光）来控制 Cas9 的激活、抑制或降解，从而实现对 Cas9 在上述维度上的精确控制。
