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TSA-超声波协同作用增强了双胞胎酵母中的CRISPR-Cas9基因编辑效率

Ge Huang¹, Bin Zhuge¹, Xueqing Du¹

¹The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Lab of Industrial Microorganism & Research and Design Center for Polyols, School of Biotechnology, Jiangnan University, Wuxi 214122, China.

Journal of microbiological methods

|March 11, 2026

概括

我们开发了一种新的CRISPR-Cas9协议,将Trichostatin A (TSA) 和超声波结合起来,以克服酵母染色素障碍. 这种方法显著提高了Candida glycerinogenes等多种类酵母中的基因编辑效率.

科学领域:

微生物工程微生物工程
合成生物学合成生物学
酵母遗传学酵母遗传学

背景情况:

克里斯普尔-Cas9对于微生物工程至关重要,但由于染色质的紧性,它在多倍体酵母中面临效率限制.
像Candida glycerinogenes这样的酵母中的紧色素存在物理障碍,阻碍了Cas9的可访问性并限制了基因操纵.
现有的方法与复杂的基因组修改在反抗性酵母菌株中的斗争.

研究的目的:

开发和验证一个标准化的协议,以提高CRISPR-Cas9在多倍体酵母中的效率.
克服Candida glycerinogenes中的基于色素的物理障碍,以改善微生物工程.
为了实现先前在工业酵母中无法实现的复杂基因改造.

主要方法:

开发了一种结合Trichostatin A (TSA) 诱导的染色质脱凝和低强度超声波的协同协议.
优化了关键参数,确定了200nm TSA (10小时) 跟着200W超声波 (3分钟) 作为最佳条件.
利用二倍体工业酵母Candida glycerinogenes作为一个模型系统.

主要成果:

与传统方法相比,该协议使单基因淘汰效率 (GPD1,TRP1) 增加了一倍以上.
成功启用了复杂的编辑,包括删除7.8kb片段和编辑11.4kb区域.
在C. glycerinogenes中证明了Cas9的可访问性和更好的编辑结果.

结论:

建立了一个简单,有效的工作流程,以克服多倍体工业酵母中的染色素障碍.
为推进基因工程和功能基因组学在反抗性酵母菌种中提供了一个实用的工具.
协同方法为微生物工程应用提供了显著的进步.

关键词:

克里斯普尔卡斯9 坎迪达糖原体 (Candida glycerinogenes) 是一种糖原体.染色体脱凝过程中的染色体脱凝通过超声波灌,使其变得更有活力.运输安全局 (TSA) 在

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