CRISPR-Cas9系统中sgRNA设计与脱靶效应评估.pdf

Hereditas (Beijing) 2015 年 11 月, 37(11): 1125 ―1136 综 述 CRISPR/Cas9 系统中sgRNA 设计与脱靶效应评估 1 2 1 1 1 1 1 谢胜松 ，张懿 ，张利生 ，李广磊 ，赵长志 ，倪攀 ，赵书红 1. 华中农业大学，农业动物遗传育种与繁殖教育部重点实验室，武汉 430070 ； 2. 中国人民解放军第161 医院妇产科，武汉 430010 摘要:基于 CRISPR/Cas9 系统介导的第三代基因组编辑技术，已成功应用于动物、植物和微生物等诸多物种 的基因组改造。如何提高 CRISPR/Cas9 技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究 热点，而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前，已有多款针 对 CRISPR/Cas9 技术的 sgRNA 设计和/或脱靶效应评估软件，但不同的软件各有优缺点。本文重点对 16 款 sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述，通过制定38 项评估指标对不同软件进行了 比较分析，最后对 11 种用于检测基因组编辑效率和脱靶的实验方法，以及如何筛选高效且特异的sgRNA 进行 了归纳总结。 关键词: CRISPR/Cas9 系统；基因组编辑；sgRNA；脱靶效应 sgRNA design for the CRISPR/Cas9 system and evaluation of its off-target effects 1 2 1 1 1 Shengsong Xie , Yi Zhang , Lisheng Zhang , Guanglei Li , Changzhi Zhao , 1 1 Pan Ni , Shuhong Zhao 1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China; 2. Department of Obstetrics and Gynecology, No.161 Hospital of PLA, Wuhan 430010, China Abstract: The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully ap- plied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful ge- nome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have ad- vantages and disadvantages respectively.