近年來(lái)廣受關(guān)注的CRISPR/Cas9系統(tǒng),憑借著其能夠通過(guò)RNA介導(dǎo),精確靶向目的DNA并修飾的能力,極大的加強(qiáng)了人們對(duì)基因工程改造的能力。自2012年Jinek等人發(fā)現(xiàn)釀膿鏈球菌中的Type Ⅱ CRISPR/Cas系統(tǒng)中的Cas9蛋白能夠通過(guò)RNA介導(dǎo)在試管中切割DNA之后的短短幾年間,CRISPR/Cas9被廣泛的運(yùn)用于DNA突變、轉(zhuǎn)錄調(diào)控干預(yù)、表觀遺傳改造、染色體位點(diǎn)標(biāo)記以及高通量的正向篩選中,取得了許多顯著的成果。豬是一種重要的農(nóng)業(yè)經(jīng)濟(jì)物種。除此之外,因?yàn)樨i與人的解剖以及生理特征上的類似性,它還是生物醫(yī)藥研究中很好的動(dòng)物模型以及臨床上異種器官移植的理想供體。因此,對(duì)豬的基因改造具有意義重大的經(jīng)濟(jì)價(jià)值、研究?jī)r(jià)值以及臨床應(yīng)用價(jià)值。我們通過(guò)對(duì)中國(guó)巴馬迷你豬的一細(xì)胞期的受精卵顯微注射Cas9mRNA與sgRNA,成功的獲得了Npc1l1敲除的個(gè)體。值得一提的是,Npc1l1位點(diǎn)的突變率達(dá)到了100%。同時(shí),我們也在豬的各種組織以及卵巢組織中檢測(cè)到了Npc1l1的突變,提示了突變能夠通過(guò)生殖細(xì)胞傳給下一代。CRISPR/Cas9的脫靶效應(yīng)是一個(gè)不容忽視的生物安全性問(wèn)題,尤其在經(jīng)濟(jì)物種... 

【文章來(lái)源】：南京大學(xué)江蘇省 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：140 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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


【參考文獻(xiàn)】：
期刊論文
[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)



本文編號(hào)：3152105