研究CRISPR/Cas9系统在体外和体内定点敲除大片段DNA
发布时间：2021-07-31 00:59
在传统的基因敲除方案中,同源重组法通常用来可以敲除一个或多个外显子。这种基因敲除方法有几个缺点,如需要胚胎干细胞、载体构建复杂、耗时且花费大、敲除片段通常较短。近年来利用锌指核酸内切酶（ZFN）、转录激活样效应因子（TALEN）和成簇间断短回文重复序列及其相关内切酶（CRISPR/Cas9）进行基因敲除,通常是利用非同源末端连接造成的移码突变使编码基因移码而失活。在哺乳动物基因组中,约80%的序列是非编码的,这些非编码的基因也被证明有重要功能。对于这些非编码基因,有时移码突变或者部分外显子的缺失并不能有效使其功能失活,只有大片段的切除基因序列才能达到敲除目的。在本研究中,我们基于CRISPR/Cas9技术建立了大片段敲除非编码基因的方法。我们首先利用Cas9蛋白在试管内验证了其切割效率,并优化了反应条件。在Cas9蛋白和sgRNA足量的情况下,用单条或2条sgRNA同时切割pEGFP-N1质粒,效率几乎都达到100%。然后我们构建了稳定表达mCherry-P2A-EGFP的293T细胞系作为报告系统,来检测CRISPR/Cas9系统在人细胞中敲除外源序列的效率。结果显示同时转染针对mC...
【文章来源】：南京大学江苏省 211工程院校 985工程院校 教育部直属院校
【文章页数】：132 页
【学位级别】：博士
【文章目录】：
Abstract
中文摘要
Abbreviations
Chapter Ⅰ A brief review Revolution in genome editing:CRISPR/Cas9 system and its applications
1.1 Gene targeting tools
1.1.1 Homologous recombination
1.1.2 Programmable nucleases
1.2 A brief introduction of CRISPR/Cas system
1.2.1 Evolution of CRISPR system
1.2.2 Components and mechanism of CRISPR/Cas system
1.2.3 Classification of CRISPR/Cas system
1.2.4 Type II CRISPR/Cas system
1.3 CRISPR/Cas9 as a genome editing tool
1.3.1 Structure of SpCas9
1.3.2 CRISPR/Cas9 in gene targeting
1.3.3 Application of Cas9 nickases
1.3.4 Application of CRISPR/Cas9 in high-throughput screening
1.3.5 Application of CRISPR/Cas9 in gene and cell therapy
1.3.6 CRISPR/Cas9 gene drives in species
1.3.7 Application of CRISPR/dCas9
1.3.8 Other representative progresses of CRISPR/Cas9
1.3.9 Strategies for improving specificity of CRISPR/Cas9
1.3.10 Comparison of CRISPR/Cas9 with ZFN and TALEN
1.4 Problems and perspectives
References
Chapter Ⅱ Efficient in vitro and in vivo cleavage of large DNA fragments by CRISPR/Cas9
Introduction
Materials and Methods
Results
2.1 CRISPR/Cas9 system for cleaving DNA in vitro
2.2 Deletion exogenous fragments in human cells by CRISPR/Cas9 system
2.3 Deletion endogenous fragments in mouse embryonic stem cells by CRISPR/Cas9 system
2.4 Efficient one-step knockout of lncRNA, Rian, via CRISPR/ Cas9-mediated fragment deletion
Discussions
References
Acknowledgements
Original publication
【参考文献】：
期刊论文
[1]Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy[J]. Jiyong Liu~a,Changqing Li~a,Zhongsheng Yu~a,Peng Huang~b,Honggang Wu~a,Chuanxian Wei~a, Nannan Zhu~a,Yan Shen~b,Yixu Chen~a,Bo Zhang~b,Wu-Min Deng~(c,*),Renjie Jiao~(a,*) a State Key Laboratory of Brain and Cognitive Science,Institute of Biophysics,The Chinese Academy of Sciences,Datun Road 15,Beijing 100101,China b Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education,College of Life Sciences,Peking University,Beijing 100871,China c Department of Biological Science,Florida State University,Tallahassee,Florida 32304-4295,USA. 遗传学报. 2012(05)
本文编号：3312448
【文章来源】：南京大学江苏省 211工程院校 985工程院校 教育部直属院校
【文章页数】：132 页
【学位级别】：博士
【文章目录】：
Abstract
中文摘要
Abbreviations
Chapter Ⅰ A brief review Revolution in genome editing:CRISPR/Cas9 system and its applications
1.1 Gene targeting tools
1.1.1 Homologous recombination
1.1.2 Programmable nucleases
1.2 A brief introduction of CRISPR/Cas system
1.2.1 Evolution of CRISPR system
1.2.2 Components and mechanism of CRISPR/Cas system
1.2.3 Classification of CRISPR/Cas system
1.2.4 Type II CRISPR/Cas system
1.3 CRISPR/Cas9 as a genome editing tool
1.3.1 Structure of SpCas9
1.3.2 CRISPR/Cas9 in gene targeting
1.3.3 Application of Cas9 nickases
1.3.4 Application of CRISPR/Cas9 in high-throughput screening
1.3.5 Application of CRISPR/Cas9 in gene and cell therapy
1.3.6 CRISPR/Cas9 gene drives in species
1.3.7 Application of CRISPR/dCas9
1.3.8 Other representative progresses of CRISPR/Cas9
1.3.9 Strategies for improving specificity of CRISPR/Cas9
1.3.10 Comparison of CRISPR/Cas9 with ZFN and TALEN
1.4 Problems and perspectives
References
Chapter Ⅱ Efficient in vitro and in vivo cleavage of large DNA fragments by CRISPR/Cas9
Introduction
Materials and Methods
Results
2.1 CRISPR/Cas9 system for cleaving DNA in vitro
2.2 Deletion exogenous fragments in human cells by CRISPR/Cas9 system
2.3 Deletion endogenous fragments in mouse embryonic stem cells by CRISPR/Cas9 system
2.4 Efficient one-step knockout of lncRNA, Rian, via CRISPR/ Cas9-mediated fragment deletion
Discussions
References
Acknowledgements
Original publication
【参考文献】：
期刊论文
[1]Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy[J]. Jiyong Liu~a,Changqing Li~a,Zhongsheng Yu~a,Peng Huang~b,Honggang Wu~a,Chuanxian Wei~a, Nannan Zhu~a,Yan Shen~b,Yixu Chen~a,Bo Zhang~b,Wu-Min Deng~(c,*),Renjie Jiao~(a,*) a State Key Laboratory of Brain and Cognitive Science,Institute of Biophysics,The Chinese Academy of Sciences,Datun Road 15,Beijing 100101,China b Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education,College of Life Sciences,Peking University,Beijing 100871,China c Department of Biological Science,Florida State University,Tallahassee,Florida 32304-4295,USA. 遗传学报. 2012(05)
本文编号：3312448
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