建立一个单步无标记CRISPR/Cas9系统用于工业Aspergillus niger,使用可反选择标记Ang-ace2
Jiao Liu1,2, Jie Zhu1, Qian Zhang1
1MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
Biotechnology letters
|October 8, 2023
概括
一个新的CRISPR/Cas9基因组编辑系统为尼日利亚虫提供高效率和减少16天的周期. 这种无标记物系统简化了工业应用中的基因改造.
科学领域:
- 微生物学 微生物学
- 分子生物学分子生物学
- 生物技术是生物技术.
背景情况:
- 工业Aspergillus niger菌株对于生物技术至关重要,但需要高效的基因组编辑工具.
- 现有的基因组编辑方法可能耗时,涉及标记基因,使菌株发展复杂化.
研究的目的:
- 开发一个单步,无标记的CRISPR/Cas9系统,用于工业Aspergillus niger的快速和高效的基因组编辑.
- 为了缩短这种工业相关的真菌中CRISPR/Cas9的基因操作周期.
主要方法:
- 评估了sgRNA表达的各种促进剂,确定tRNAGly15以获得最佳的效率.
- 开发了一种CRISPR/Cas9等离子体,其中包含一个截断的AMA1元素和Ang-ace2反选择性标记物.
- 使用Tet-on促进剂对Ang-ace2进行条件表达,从而实现无标记物选择.
主要成果:
- 使用tRNAGly15促进器实现100%的基因编辑效率,用于sgRNA表达.
- 在A. niger后代中,经过一代的多西环林诱导后,证明了33%的标记物损失效率.
- 成功地将CRISPR/Cas9基因操作周期缩短到16天.
结论:
- 一个单步,无标记的CRISPR/Cas9系统已经成功地在工业Aspergillus niger.中建立.
- 该系统使得高效的基因组编辑能够在显著缩短的时间内实现.
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