Brief Funct Genomics. 2017 Jul 1;16(4):211-216. doi: 10.1093/bfgp/elw038.
The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system is a recently discovered tool for genome editing that has quickly revolutionized the ability to generate site-specific mutations in a wide range of animal models, including nonhuman primates. Indeed, a significant number of scientific reports describing single or multiplex guide RNA microinjection, double-nicking strategies, site-specific knock-in and conditional knock-out have been published in less than three years. However, despite the great potential of this new technology, there are some limitations because of the presence of off-target genomic sites, which must be taken into consideration. To address this issue, various research teams have tried to improve the efficiency of the system through enzymatic modifications of the Cas9 protein or by the introduction of alternative strategies. Although several review articles are available that singly describe the molecular mechanism(s), applications and challenges of each of these strategies, a concise compilation of approaches is lacking. In the current review, we describe and evaluate most CRISPR/Cas9 approaches available at present, describing both mechanism of action, in addition to advantages or disadvantages. The primary goal of this work is to serve as a guide for not skilled researchers, facilitating the selection of the best strategy to target their gene of interest and allowing optimization of particular applications to the specific aims of the study. The present article also offers a unique perspective, focusing on the fact that CRISPR technology is opening a new genomic era, providing the means to manipulate specific genes in a targeted manner in all animal models, an endeavor previously considered to be difficult.
成簇规律间隔短回文重复（CRISPR/Cas9）系统是一种新发现的基因组编辑工具，它迅速改变了在广泛的动物模型（包括非人类灵长类动物）中产生特定位置突变的能力。事实上，在不到三年的时间里，已经发表了大量描述单或多导 RNA 微注射、双切口策略、特异性敲入和条件敲除的科学报告。然而，尽管这项新技术具有很大的潜力，但由于存在脱靶基因组位点，因此存在一些限制，必须加以考虑。为了解决这个问题，许多研究小组试图通过 Cas9 蛋白的酶修饰或引入替代策略来提高系统的效率。尽管有几篇综述文章分别描述了这些策略中的每一种的分子机制、应用和挑战，但缺乏简洁的方法汇编。在目前的综述中，我们描述和评估了目前可用的大多数 CRISPR/Cas9 方法，描述了作用机制，以及优点和缺点。这项工作的主要目标是为非熟练研究人员提供指导，帮助他们选择针对感兴趣基因的最佳策略，并优化特定应用以满足研究的具体目标。本文还提供了一个独特的视角，重点关注 CRISPR 技术正在开启一个新的基因组时代，提供了有针对性地操纵所有动物模型中特定基因的手段，这在以前被认为是困难的。
