| 李琦,安成泽,路文欣,等.南极海洋假裸囊属真菌CRISPR-Cas9敲除体系构建[J].中国海洋药物,2026,(4):-. |
| 南极海洋假裸囊属真菌CRISPR-Cas9敲除体系构建 |
| Development of a CRISPR-Cas9 Knockout System for Antarctic Marine-Derived Pseudogymnoascus Fungi |
| 投稿时间：2025-04-11 修订日期：2025-05-23 |
| DOI： |
| 中文关键词: 南极真菌 CRISPR-Cas9 假裸囊菌属 |
| English Keywords:Antarctic fungi CRISPR-Cas9 Pseudogymnoascus |
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| 中文摘要: |
| 目的 构建南极海洋沉积物来源假裸囊属真菌CRISPR-Cas9敲除体系。方法 使用三种启动子启动两种来源的Cas9蛋白表达,通过RNA提取在转录层面验证蛋白的表达,通过荧光指示观察蛋白的表达情况,选择Cas9表达成功的菌株进行原生质体制备。通过In-Fusion无缝克隆技术构建sgRNA表达载体,通过T7启动子启动sgRNA体外转录从而提供两种敲除方案。结果 在构建出的6个蛋白表达质粒中PtrpC启动子在OUCMDZ-3597中成功启动Cas9-334蛋白的表达,在转录水平得以验证,并通过荧光蛋白指示与野生株对比,观测到Cas9蛋白的表达。成功构建两个sgRNA表达通用质粒,一个可用于插入敲除供体DNA片段、完成sgRNA靶序列的替换并带有潮霉素B抗性,另一个可用于sgRNA特异性扩增。体外转录的方式作为sgRNA表达载体在宿主中无法正常启动的替代方案,为假裸囊属真菌CRISPR-Cas9提供技术支持。 |
| English Summary: |
| Objective To develop a CRISPR-Cas9 knockout system of Pseudogymnoascus fungi from Antarctic marine sediments. Methods Three kinds of promoters were used to activate the expression of Cas9 protein from two sources. Protein expression was verified at the transcriptional level by RNA extraction and was observed by fluorescence detection. The Cas9 strains with successfully expressed were selected for protoplasm preparation. The expression vector of sgRNA was constructed by In-fusion seamless cloning technology. The in vitro transcription of sgRNA was driven by the T7 promoter. Ultimately two strategies of the knockout system were provided. Results In the six protein expression plasmids constructed, the PtrpC promoter successfully activated the expression of Cas9-334 protein in OUCMDZ-3597, which was verified at the transcriptional level. Through fluorescent protein indication and comparison with wild-type strains, the expression of Cas9 protein was observed. Two universal sgRNA expression plasmids were successfully constructed. One enables the insertion of knockout donor DNA fragments, the replacement of sgRNA target sequences, and carries hygromycin B resistance; the other facilitates specific amplification of sgRNA. Using in vitro transcription as an alternative approach when sgRNA expression vectors fail to initiate properly in the host. This study provides technical support for CRISPR-Cas9 applications in Pseudogymnoascus fungi. |
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