基于Meganucleases,ZFNs,TALENs,和CRISPR/Cas9系統(tǒng)的基因編輯工具已經(jīng)在大量細胞系以及器官中應(yīng)用于精確的基因修飾。與以前的基因編輯工具相比較,最近興起的CRISPR/Cas9基因編輯系統(tǒng)由于其簡便性、可定制性以及多功能性,展現(xiàn)出了卓越的優(yōu)勢。最普遍的CRISPR/Cas9基因編輯系統(tǒng)主要由兩個元件構(gòu)成:Cas9核酸酶以及導(dǎo)向RNA。CRISPR/Cas9基因編輯系統(tǒng)在很多領(lǐng)域被寄予厚望,包括基因功能研究,基因治療,異種器官移植以及動物繁育。很多研究也都在試圖解決該系統(tǒng)的瓶頸與挑戰(zhàn)。其中,對與靶位點的選擇并且產(chǎn)生有生物活性的導(dǎo)向RNA對于該系統(tǒng)的效率以及靶向編輯的精確性具有重大意義,目前已經(jīng)有許多對于增強靶向單個位點或多個位點的導(dǎo)向RNA的表達量的嘗試研究。細菌的T7 RNA聚合酶是一個活性很高并且特異性依賴于其自身T7啟動子的RNA聚合酶。酵母,尤其是釀酒酵母,被視為研究很廣泛的單細胞真核模式生物。在本實驗室,應(yīng)用T7 RNA聚合酶/T7啟動子這套系統(tǒng),通過PCR技術(shù),在酵母細胞中表達有活性的導(dǎo)向RNA用來指導(dǎo)Cas9進行靶位點的編輯。sgRNA的功能是... 

【文章來源】：西北農(nóng)林科技大學陜西省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：82 頁

【學位級別】：碩士

【文章目錄】：
abstract
摘要
Acknowledgements
Introduction
Chapter 1:Literature review
    1.1 An overview of genetic modification
    1.2 Gene-editing nucleases:from meganucleases to CRISPR/Cas systems
        1.2.1 Meganucleases
        1.2.2 Zinc finger nucleases （ZFNs）
        1.2.3 Transcription activator-like effector nucleases （TALENs）
        1.2.4 Clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated proteins （Cas）
    1.3 A glimpse on CRISPR systems: from observed short repetitions to versatile genome editing tools
    1.4 Applications of CRISPR/Cas system in yeast
    1.5 Applications of CRISPR/Cas system in animal
        1.5.1 Sheep
        1.5.2 Goats
        1.5.3 Cattle
        1.5.4 Pigs
        1.5.5 Rabbits
        1.5.6 Chickens
Chapter 2:Using T7-sgRNAs to evaluate the efficiencies of different target sites in yeast
    2.1 Materials and methods
        2.1.1 Strains, plasmids, and media
        2.1.2 Generation of linear T7-sgRNAs
            2.1.2.1 Selection of target sites
            2.1.2.2 Primer design
            2.1.2.3 PCR program
            2.1.2.4 Gel electrophoresis and gel extraction
        2.1.3 Yeast transformation
    2.2 Results
    2.3 Discussion
    2.4 Summary
References
Appendices
About the author
Publications


【參考文獻】：
期刊論文
[1]Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts[J]. LIU Hui,LIU Chang,ZHAO Yu-hang,HAN Xue-jie,ZHOU Zheng-wei,WANG Chen,LI Rong-feng,LI Xue-ling.  Journal of Integrative Agriculture. 2018(02)
[2]Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting[J]. Ze Li,Hai-Yuan Yang,Ying Wang,Man-Ling Zhang,Xiao-Rui Liu,Qiang Xiong,Li-Ning Zhang,Yoong Jin,Li-Sha Mou,Yan Liu,Rong-Feng Li,Yi Rao,Yi-Fan Dai.  The Journal of Biomedical Research. 2017(05)
[3]Advances in genetic engineering of domestic animals[J]. Shaohua WANG,Kun ZHANG,Yunping DAI.  Frontiers of Agricultural Science and Engineering. 2016(01)



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