Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
Department of Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht 4199613776, Iran.
Int J Mol Sci. 2019 Jul 30;20(15):3719. doi: 10.3390/ijms20153719.
The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.
CRISPR/Cas9 系统（成簇规律间隔短回文重复相关蛋白 9）是一种在动物、植物和人类中强大的基因组编辑工具。与传统的基因组编辑工具相比，如 meganuclease、转录激活因子样效应物核酸酶（TALENs）和锌指核酸酶（ZFNs），该系统具有一些优势，例如高靶向突变率（靶向效率）、成本低、简单、高效的多位点编辑。该系统的一个关键缺点是在脱靶位点产生非预期的突变。我们总结和讨论了不同的方法，如 dCas9 和 Cas9 配对切口酶，以减少植物中的脱靶效应。根据研究，减少非预期突变的最有效方法是使用称为适体酶的配体依赖性核酶。单指导 RNA（sgRNA）/配体依赖性适体酶策略帮助研究人员避免了人类细胞中的非预期突变，并且可以作为一种替代方法用于植物，以显著降低脱靶突变的频率。我们希望我们的概念为基因转化和基因组编辑提供一种新的、简单和快速的方法，具有减少时间和能源消耗、避免非预期突变、增加靶向变化频率以及无需外部力量或昂贵设备的优点。
