在傳統(tǒng)的基因敲除方案中,同源重組法通常用來(lái)可以敲除一個(gè)或多個(gè)外顯子。這種基因敲除方法有幾個(gè)缺點(diǎn),如需要胚胎干細(xì)胞、載體構(gòu)建復(fù)雜、耗時(shí)且花費(fèi)大、敲除片段通常較短。近年來(lái)利用鋅指核酸內(nèi)切酶（ZFN）、轉(zhuǎn)錄激活樣效應(yīng)因子（TALEN）和成簇間斷短回文重復(fù)序列及其相關(guān)內(nèi)切酶（CRISPR/Cas9）進(jìn)行基因敲除,通常是利用非同源末端連接造成的移碼突變使編碼基因移碼而失活。在哺乳動(dòng)物基因組中,約80%的序列是非編碼的,這些非編碼的基因也被證明有重要功能。對(duì)于這些非編碼基因,有時(shí)移碼突變或者部分外顯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達(dá)到敲除目的。在本研究中,我們基于CRISPR/Cas9技術(shù)建立了大片段敲除非編碼基因的方法。我們首先利用Cas9蛋白在試管內(nèi)驗(yàn)證了其切割效率,并優(yōu)化了反應(yīng)條件。在Cas9蛋白和sgRNA足量的情況下,用單條或2條sgRNA同時(shí)切割pEGFP-N1質(zhì)粒,效率幾乎都達(dá)到100%。然后我們構(gòu)建了穩(wěn)定表達(dá)mCherry-P2A-EGFP的293T細(xì)胞系作為報(bào)告系統(tǒng),來(lái)檢測(cè)CRISPR/Cas9系統(tǒng)在人細(xì)胞中敲除外源序列的效率。結(jié)果顯示同時(shí)轉(zhuǎn)染針對(duì)mC... 

【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)】：132 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 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


【參考文獻(xiàn)】：
期刊論文
[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.  遺傳學(xué)報(bào). 2012(05)



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