National Center for Clinical Laboratories, Beijing Hospital, Beijing, China.
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
FEBS J. 2016 Apr;283(7):1218-31. doi: 10.1111/febs.13586. Epub 2015 Nov 27.
Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.
最近，一种名为成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白（Cas）9 系统的新型技术得到了迅速发展。这种基因组编辑工具极大地提高了我们探索疾病发病机制和纠正疾病突变以及表型的能力。通过短向导 RNA，Cas9 可以精确地靶向目标位点，并作为内切酶有效地在 DNA 双链中产生断裂。在过去的 30 年中，CRISPR 已经从“具有未知生物学功能的好奇序列”演变成了一种有前途的基因组编辑工具。由于 CRISPR/Cas9 系统的不断发展，与定制向导 RNA 共表达的 Cas9 已成功用于多种细胞和生物体。这种基因组编辑技术也可应用于合成生物学、功能基因组筛选、转录调控和基因治疗。然而，尽管 CRISPR/Cas9 在科学上具有广泛的作用，但有几个方面会影响其效率和特异性，包括 Cas9 活性、靶位点选择和短向导 RNA 设计、递送方法、脱靶效应和同源定向修复的发生率。在本综述中，我们重点介绍了影响 CRISPR/Cas9 利用的因素，以及处理任何问题的可能策略。解决这些问题将使我们能够更好地利用这项技术。此外，我们还回顾了 CRISPR/Cas 系统从 2012 年首次发现以来的历史和快速发展。
