在Saccharomyces cerevisiae中改进了用于反子介导的CRISPR-Cas9基因组编辑的载体
Tara N Stuecker1, Stephanie E Hood1, Julio Molina Pineda1,2
1Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas, United States of America.
bioRxiv : the preprint server for biology
|August 16, 2024
概括
我们开发了一个精简的基于CRISPR的系统,用于在酵母中精确的基因编辑. 这种方法简化了局部定向的突变发生,在各种酵母菌株中实现了点突变和删除的高编辑效率.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 酵母生物学的酵母生物学
背景情况:
- 位点导向的突变发生对于了解体内基因功能至关重要.
- 克里斯普尔-Cas9系统提供高效的基因编辑,但在原生酵母菌株方面存在局限性.
- 目前的方法通常涉及指导RNA (gRNA) 的低效克隆.
研究的目的:
- 开发一种基于CRISPR的简化和高效的系统,用于Saccharomyces cerevisiae的局部定向突变发生.
- 克服现有方法在完全原性酵母菌株的局限性.
- 集成gRNA和修复模板设计,以简化编辑.
主要方法:
- Cas9和大肠杆菌逆子 (EcRT) 的联合表达用于在体内放大修复模板作为多副本单链DNA (msDNA).
- 组合gRNA和修复模板的单个寡核酸的设计,用于cis-acting编辑.
- 开发一种基于等离子体的系统,具有多种可选择的标记物 (抗生素和辅助性) 和可诱导的促进物 (银糖和β-雌激素).
主要成果:
- 实现了>95%的点突变编辑效率和>50%的无标记删除.
- 在完全原性野生酵母菌株中成功编辑.
- 通过beta-estradiol诱导,使酵母菌株具有较差的银河糖利用率实现了高效的编辑.
结论:
- 开发的基于等离子体的系统显著简化并提高了酵母中CRISPR介导的局部定向突变发生的效率.
- 这种方法是多用途的,适用于各种酵母菌株,并减少了基因编辑所需的时间和步骤.
- 为研究人员提供了一个详细的协议,以实现这种先进的基因编辑工具.
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