目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6...目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6J小鼠,针对miR-551b外显子1区域,设计导向RNA(guide RNA,gRNA),构建Cas9载体质粒,将体外转录的Cas9 RNA及gRNA显微注射入小鼠的受精卵并体外培养。将培养合格的胚胎移植到代孕小鼠的输卵管中,待小鼠生育后得到F0代小鼠,使用基因测序确定基因敲除情况,与野生型小鼠繁育后,得到F1代杂合小鼠,F1代小鼠经自交繁育获得F2代小鼠,F3代小鼠由F2代纯合小鼠自交获得,采用电泳鉴定小鼠基因型,RT-PCR检测F3代小鼠组织miR-551b的表达。结果:利用CRISPR/Cas9技术构建模型小鼠得到F0代小鼠,通过测序筛选出缺失目标序列的F0代杂合子小鼠。与WT小鼠繁育后,琼脂糖凝胶电泳及测序筛选出F1代杂合小鼠,同样的方法鉴定并获得F2、F3代基因敲除小鼠,获取F3代纯合小鼠的心脏及下腔静脉样本,RT-PCR结果证实F3代纯合小鼠miR551b表达明显低于WT小鼠(P<0.05),成功敲除miR-551b基因。结论:通过CRISPR-Cas9技术成功构建miR⁃155基因敲除小鼠模型并稳定遗传,为进一步研究提供了有利条件。展开更多
从原核生物的适应性免疫机制衍生出的成簇规律间隔短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeat and associated protein,CRISPR-Cas)系统,以其特异性高、可开发性强、简单高效等特点迅速从...从原核生物的适应性免疫机制衍生出的成簇规律间隔短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeat and associated protein,CRISPR-Cas)系统,以其特异性高、可开发性强、简单高效等特点迅速从众多具有竞争力的基因编辑技术中脱颖而出,既为体内基因编辑技术带来革命性突破,也在体外诊断领域开拓了新的方向。近年来涌现出许多快速、便携、经济、高效的基于CRISPR-Cas系统的体外诊断技术,这些技术在病原体检测方面展示出良好的性能,在肿瘤基因诊断、遗传病筛查、移植排斥反应检测等方面也具有很大潜力。此文仅就CRISPR-Cas系统的作用原理、基于CRISPR-Cas系统的检测方法以及在病原体核酸检测领域的应用进展进行简述。展开更多
This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequen...This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.展开更多
文摘从原核生物的适应性免疫机制衍生出的成簇规律间隔短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeat and associated protein,CRISPR-Cas)系统,以其特异性高、可开发性强、简单高效等特点迅速从众多具有竞争力的基因编辑技术中脱颖而出,既为体内基因编辑技术带来革命性突破,也在体外诊断领域开拓了新的方向。近年来涌现出许多快速、便携、经济、高效的基于CRISPR-Cas系统的体外诊断技术,这些技术在病原体检测方面展示出良好的性能,在肿瘤基因诊断、遗传病筛查、移植排斥反应检测等方面也具有很大潜力。此文仅就CRISPR-Cas系统的作用原理、基于CRISPR-Cas系统的检测方法以及在病原体核酸检测领域的应用进展进行简述。
文摘This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.
