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基于CRISPR-Cas9系统的Saccharomyces cerevisiae基因删除

2020-12-02阅读(805)

基于CRISPR-Cas9系统的Saccharomyces cerevisiae基因删除

论文摘要

建立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 d。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 d,基因编辑效率分别为9/32和10/32。

论文目录

  • 1 材料与方法
  •   1.1?材料与试剂
  •     1.1.1?菌株与质粒
  •     1.1.2 引物与试剂?
  •     1.1.3 培养基
  •   1.2 仪器与设备
  •   1.3 方法
  •     1.3.1 菌株培养
  •     1.3.2 CRISPR-Cas9系统的构建
  •     1.3.3 S.cerevisiae感受态的制备及转化
  •     1.3.4 can1靶点的筛选验证
  •     1.3.5 其他基因敲除的筛选验证
  •   1.4 数据及图像处理
  • 2 结果与分析
  •   2.1 Cas9系统的构建
  •     2.1.1 pRS424-TRP-cas9质粒的构建
  •     2.1.2 can1基因gRNA打靶质粒的构建
  •   2.2 can1靶点基因敲除的结果
  •   2.3 其他单基因敲除的结果
  •   2.4 基因连续敲除的结果
  •   2.5 双基因敲除的筛选验证结果
  • 3 讨论

    • 文章来源

    类型: 期刊论文

    作者: 牛潇迪,李天明,刘金雷,杨天勇,李子怡,冯惠勇

    关键词: 酿酒酵母,基因组工程,基因敲除

    来源: 食品科学 2019年06期

    年度: 2019

    分类: 工程科技Ⅰ辑,基础科学

    专业: 生物学

    单位: 河北科技大学生物科学与工程学院,马里兰大学帕克分校文理科学学院

    基金: “十二五”农村领域国家科技计划项目(2015BAD15B0501)

    分类号: Q78

    页码: 151-158

    总页数: 8

    文件大小: 3769K

    下载量: 258

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    基于CRISPR-Cas9系统的Saccharomyces cerevisiae基因删除
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