利用CRISPR/Cas9系统建立基因修饰猪以及在人细胞中对顺式作用元件做注释

发布时间：2021-04-21 16:35

　　近年来广受关注的CRISPR/Cas9系统,凭借着其能够通过RNA介导,精确靶向目的DNA并修饰的能力,极大的加强了人们对基因工程改造的能力。自2012年Jinek等人发现酿脓链球菌中的Type Ⅱ CRISPR/Cas系统中的Cas9蛋白能够通过RNA介导在试管中切割DNA之后的短短几年间,CRISPR/Cas9被广泛的运用于DNA突变、转录调控干预、表观遗传改造、染色体位点标记以及高通量的正向筛选中,取得了许多显著的成果。猪是一种重要的农业经济物种。除此之外,因为猪与人的解剖以及生理特征上的类似性,它还是生物医药研究中很好的动物模型以及临床上异种器官移植的理想供体。因此,对猪的基因改造具有意义重大的经济价值、研究价值以及临床应用价值。我们通过对中国巴马迷你猪的一细胞期的受精卵显微注射Cas9mRNA与sgRNA,成功的获得了Npc1l1敲除的个体。值得一提的是,Npc1l1位点的突变率达到了100%。同时,我们也在猪的各种组织以及卵巢组织中检测到了Npc1l1的突变,提示了突变能够通过生殖细胞传给下一代。CRISPR/Cas9的脱靶效应是一个不容忽视的生物安全性问题,尤其在经济物种... 

【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】：140 页

【学位级别】：博士

【文章目录】：
Abstract
中文摘要
Abbreviations
Chapter Ⅰ A brief review about CRISPR/Cas9 system and its applications as abiotechnology tool
    1.1 Background of CRISPR/Cas systems
        1.1.1 History of CRISPR/Cas research
        1.1.2 Common features of CRISPR/Cas systems
        1.1.3 Classification of CRISPR/Cas systems
        1.1.4 Milestone of CRISPR/Cas9 research
    1.2 Principles and methodology for genome editing by CRISPR/Cas9
        1.2.1 Principle for KO and KI by Cas9 nucleases
        1.2.2 Principle for genome targeting and modification by dead Cas9
        1.2.3 Methods to enhance genome editing specificity of CRISPR/Cas9
        1.2.4 Methods to increase efficiency of HDR
        1.2.5 Comparison of ZFNs, TALENs and CRISPR/Cas9 systems
    1.3 Applications of CRISPR/Cas9
        1.3.1 KO or KI by Cas9 nucleases in diverse species
        1.3.2 Somatic genome editing by Cas9 nucleases for diseases modeling
        1.3.3 Preclinical trials through KO endogenous genes or viral genomes by Cas9 nucleases
        1.3.4 Gene regulation by dCas9
    1.4 Summary and perspective
    References
Chapter Ⅱ Efficient generation of gene-modified pigs via injection of zygote withCas9/sgRNA and TALENs
    2.1 Abstract
    2.2 Introduction
    2.3 Efficient generation of Npc1l1-null pigs via injection of zygote with Cas9/sgRNA
        2.3.1 Introduction
        2.3.2 Materials and methods
        2.3.3 Results
        2.3.4 Discussion
    2.4 Efficient generation of B2m-null pigs via injection of zygote with TALENs
        2.4.1 Introduction
        2.4.2 Materials and methods
        2.4.3 Results
        2.4.4 Discussion
    2.5 Summary and discussion
    References
Chapter Ⅲ Functional annotation of cis-regulatory elements in human cells bydCas9/sgRNA
    3.1 Abstract
    3.2 Introduction
    3.3 Materials and methods
        3.3.1 Reagents
        3.3.2 Vector constructs
        3.3.3 Antibodies
        3.3.4 Cell culture and transfection
        3.3.5 T7EN1 cleavage assay
        3.3.6 Luciferase activity assay
        3.3.7 RT-qPCR assay
        3.3.8 Chromatin Immuno-precipitation（ChIP）analyses
    3.4 Results
        3.4.1 Cis-element-specific dCas9/sgRNA works effectively against a reporter construct
        3.4.2 dCas9/sgRNA can effectively target endogenous cis-elements with minimal efforts of optimization
        3.4.3 dCas9/sgRNA can independently target cis-elements separated by short spacers and can confer multiplexed targeting
        3.4.4 dCas9/sgRNA-mediated cis-element targeting may proceed with considerable specificity
    3.5 Discussion
    References
Acknowledgements
Publications


【参考文献】：
期刊论文
[1]Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing[J]. Yangbin Gao,Yunde Zhao.  Journal of Integrative Plant Biology. 2014(04)
[2]Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer[J]. BAO Lei,CHEN HaiDe,JONG UiMyong,RIM CholHo,LI WenLing,LIN XiJuan,ZHANG Dan,LUO Qiong,CUI Chun,HUANG HeFeng,ZHANG Yan,XIAO Lei,FU ZhiXin.  Science China（Life Sciences）. 2014(02)



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