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• 生物工程 •    下一篇

基于CRISPR-Cas9系统的Saccharomyces cerevisiae基因删除研究

牛潇迪1,李天明2,刘金雷2,杨天勇1,李子怡3,冯惠勇2   

  1. 1. 河北科技大学研究生学院
    2. 河北科技大学生物科学与工程学院
    3. University of Maryland, College Park
  • 收稿日期:2018-01-24 修回日期:2019-01-23 出版日期:2019-03-25 发布日期:2019-04-02
  • 通讯作者: 冯惠勇 E-mail:fenghuiyong@163.com
  • 基金资助:
    国家支撑计划项目

Research of Saccharomyces cerevisiae Gene Deletion Based on CRISPR-Cas9 System

Xiao-Di NIU 2, 2, 2,   

  • Received:2018-01-24 Revised:2019-01-23 Online:2019-03-25 Published:2019-04-02

摘要: 本论文建立了CRISPR-Cas9 介导的在Saccharomyces cerevisiae双倍体细胞中进行基因敲除的方法。以can1基因敲除后的表型,验证了该CRISPR-Cas9系统的有效性, can1基因的失活效率达到4%。利用该系统又分别敲除了pdc、adh3、adh2、adh1、 pdh等基因,单基因编辑效率分别为4/48、3/48、1/48、3/28、1/16。确定了基因连续敲除的方法流程,pdc、adh3、adh2三个基因全部敲除,整个过程用时17天。探索了双基因一次转化同时敲除的方法,将adh5、lip两个基因同时敲除用时6天,基因编辑效率分别为9/32和10/32

关键词: 酿酒酵母, CRISPR-Cas9, 基因组工程, 基因敲除

Abstract: In this dissertation, the CRISPR-Cas9 system was established in Saccharomyces cerevisiae, and a method of gene knockout in diploid cells was reported using this gene editing technique.The experimental results shows that the inactivation efficiency of can1 gene reaches 4%. The genes of pdc, adh3, adh2, adh1 and pdh were knocked out by this system respectively, and the efficiency of gene editing was 4/48, 3/48, 1/48, 3/28 and 1/16, respectively. This article also identified the method for continuous knockout gene, we completely knocked out pdc, adh3, adh2 three genes in 17 days . As proof of concept, two genes, adh5 and lip were simultaneously disrupted in 6 days with 9/32 and 10/32 efficiency respectively.

Key words: Saccharomyces cerevisiae, CRISPR-Cas9, Genome Engineering, Gene Knockout

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