论文总字数：19240字

摘 要

长久以来，科学家们一直在努力研究对基因组在单核苷酸水平上进行精确修改，这对遗传性应用，生物技术，药物研发都有很大的帮助。但是，对模型生物和人类细胞操作的相关技术却有时间长，操作复杂，效率低，容易脱靶等缺陷。CRISPR系统操作简单而且成功率高，在靶向基因敲除工具中很突出。与ZFN/TALEN相比，CRISPR/Cas更易于操作，效率更高，更容易得到纯合子突变体，而且可以在不同的位点同时引入多个突变。但该系统是否有脱靶效应尚需进一步的研究。

本设计以大肠杆菌为对象，选择一个lacZ基因，分析大肠杆菌乳糖操纵子基因启动子序列并设计靶向乳糖操纵子基因启动子序列的gRNA序列，构建相关gRNA载体，转化大肠杆菌感受态细胞，然后培养细菌，筛选抗生素药物得到候选克隆，用PCR、测序及lacZ酶活性测定分析候选克隆，探讨CRISPR/Cas9系统用于敲除大肠杆菌基因的实验方案。

关键词：CRISPR/Cas系统；基因敲除；lacZ基因；大肠杆菌

CRISPR / Cas9 system for E. coli lacZ gene knockout

ABSTRACT

For a long time, scientists have been trying to study the precise modification of the genome in a single nucleotide level, it is helpful for hereditary application, biotechnology, pharmaceutical development . However, the model of human cell biology and related technologies operating have some defects，such as need a long time, complex operation, low efficiency, easy to miss. CRISPR system is simple and the success rate is high, the targeted gene knockout tool is very prominent. Compared with the ZFN / TALEN, CRISPR / Cas easier to operate, more efficient and easier to obtain homozygous mutants, and can simultaneously introduce multiple mutations at different loci. However, the system need to study further off-target effects.

The design of E. coli as an object, select a lacZ gene, analysis of E. coli lactose operon promoter sequences designed to target the lac operon gene promoter sequence gRNA sequence, build relevant gRNA vector,transformed into E. coli competent cells,then culture of bacteria,screening of antibiotics and obtained candidate clone.By PCR, sequencing and analysis of lacZ activity assay candidate clones,discussion CRISPR / Cas9 system pilot program for knockout E. coli genes.

Keywords: CRISPR / Cas systems; knockout; lacZ gene; E. coli

目 录

摘要·······································································II

第一章 文献综述··························································III

1.1 课题研究背景························································2

1.1.1 基因敲除······················································2

1.1.2 TALENs和ZFN技术··············································2

1.1.3 CRISPR/Cas9系统···············································3

1.1.4 CRISPR/Cas9系统的结构·········································4

1.1.5 CRISPR/Cas9系统的3个主要类型·································5

1.1.6 CRISPR/Cas9系统主要类型作用机制及Cas 蛋白的多样性············6

1.1.7 CRISPR/Cas9系统的技术优势·····································7

1.2 CRISPR/Cas9系统的研究进展···········································7

1.3 本课题的研究目的与意义··············································7

第二章 材料与方法··························································7

2.1 实验材料·····························································7

2.2 实验方法·····························································9

第三章 结果与分析·························································14

参考文献···································································17

第一章 文献综述

1.1 课题研究背景

1.1.1 基因敲除

基因敲除(knockout)是指一种遗传工程技术，针对某个序列已知但功能未知的序列，改变生物的遗传基因，令特定的基因功能丧失作用，从而使部分功能被屏障，并可进一步对生物体造成影响，进而推测出该基因的生物学功能。基因敲除和基因嵌入技术是上个世纪90年代出现的最新外源DNA导入技术。基因敲除是基因打靶技术的一种，类似于基因的同源重组。指外源DNA与受体细胞基因组中序列相同或相近的基因发生同源重组，从而代替受体细胞基因组中的相同/相似的基因序列，整合入受体细胞的基因组中。此法可产生精确的基因突变，也可正确纠正机体的基因突变。基因嵌入又称基因置换，它是利用内源基因序列两侧或外面的断裂点，用同源序列的目的基因整个置换内源基因。功能基因组学的研究进展使基因敲除技术显得尤为重要。基因敲除技术从载体构建到细胞的筛选到动物模型的建立各方面都得到了发展,此外,进退策略、双置换法、标记和交换法、重组酶介导的盒子交换法也从不同方向发展了基因敲除技术^[1]。

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