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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (1): 71-76    DOI: 10.13523/j.cb.20190109
综述     
基于体外组装核糖核蛋白形式的CRISPR/Cas9基因编辑方法研究进展 *
潘海峰,杨晗,于思远,李廷栋,葛胜祥()
厦门大学分子疫苗学与分子诊断学国家重点实验室 厦门大学国家传染病诊断试剂与疫苗工程技术研究中心 厦门大学公共卫生学院 厦门 361005
Progress in Gene Editing Methods of CRISPR/Cas9 Based on in Vitro Assembly of Ribonucleoprotein
Hai-feng PAN,Han YANG,Si-yuan YU,Ting-dong LI,Sheng-xiang GE()
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen 361005,China
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摘要：

CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated)系统是近年来发展起来的新型的基因编辑技术,在生物医学领域得到广泛应用。CRISPR/Cas9系统需要在gRNA存在的条件下通过Cas9蛋白实现对基因组的定点编辑,通常情况下以慢病毒感染或质粒转染等方式提供Cas9和gRNA。但是,这些方式容易引起免疫反应及基因片段不可控插入,存在一定的风险,限制了CRISPR/Cas9技术在机体的应用。近年来发展起来的基于体外组装的核糖核蛋白(ribonucleoprotein, RNP)转导入胞的策略由于快捷安全、编辑的脱靶率低等优势引起广泛关注。对Cas9 RNP的转导方式及其应用进行了总结,并就其目前存在的问题进行探讨,以期为CRISPR/Cas9技术的进一步发展提供依据,为拓展其应用奠定基础。
关键词： CRISPR/Cas9;  核糖核蛋白;  CPP-RNP    
Abstract:

CRISPR (clustered ordered interspaced short palindromic repeats)/Cas (CRISPR-associated) system is a novel gene editing technology developed in recent years, which is widely used in the biomedical fields. The Cas9 protein and gRNA is essential for the site-specific editing of the genome by CRISPR/Cas9 system. Usually, Cas9 and gRNA are provided by lentivirus infection or plasmid transfection. However, these approaches are likely to cause adverse effects such as immune reactions and uncontrolled insertion of gene fragments, and limits the application of CRISPR/Cas9 systems. The strategy of RNP transduction based on in vitro assembly of Cas9 and gRNA developed in recent years has attracted wide attention because of its advantages such as rapid, safe and low off-target effect. The way of transduction of Cas9 RNP and its application are summarized, and its current problems are discussed in order to provide basis for the further development of CRISPR/Cas9 technology and lay a foundation for its application.
Key words: CRISPR/Cas9    Ribonucleoprotein    CPP-RNP
收稿日期: 2018-08-20 出版日期: 2019-02-28
ZTFLH:  Q78  
基金资助: * 厦门市重大科技专项资助项目(3502Z20171001-20170302)
通讯作者: 葛胜祥     E-mail: sxge@xmu.edu.cn
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潘海峰
杨晗
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葛胜祥

引用本文:

潘海峰,杨晗,于思远,李廷栋,葛胜祥. 基于体外组装核糖核蛋白形式的CRISPR/Cas9基因编辑方法研究进展 *[J]. 中国生物工程杂志, 2019, 39(1): 71-76.

Hai-feng PAN,Han YANG,Si-yuan YU,Ting-dong LI,Sheng-xiang GE. Progress in Gene Editing Methods of CRISPR/Cas9 Based on in Vitro Assembly of Ribonucleoprotein. China Biotechnology, 2019, 39(1): 71-76.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190109    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I1/71

图1  Cas9 RNP的组装及其作用机制
项目 质粒/病毒 体外转录Cas9 mRNA及gRNA RNP 参考文献
编辑效率 ++ ++ +++ [19]
插入突变 +++ - - [13]
免疫性 +++ ++ + [14]
脱靶率 +++ ++ + [18]
组分胞内表达时间 >12h >2h - [18]
组分稳定性 +++ + ++ [15,17]
表1  CRISPR/Cas9不同形式下基因编辑的比较
项目 物理转导 化学转导 纳米载体介导 细胞穿膜肽介导 参考文献
生理伤害 高 中 中 低 [24,40]
主动靶向性 无 无或弱 弱 中 [19,40-41]
借助仪器 是 否 否 否 [40]
转染试剂使用 无或需要 需要 无或需要 无 [40]
转导效率 高 中 中 中 [19,31]
血清耐受性 中 低 中 低 [19]
细胞兼容性 强 中 中 强 [39]
表2  RNP不同转导方式特点比较
图2  CPP-Cas9 RNP复合物入胞实现基因编辑示意图
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