在Streptomyces中开发一个CRISPR/Cas9D10A尼克酶 (nCas9) 中介的基因组编辑工具
Jia-Xiang Ma1, Wen-Yan He1, Hui-Min Hua1
1College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.
ACS synthetic biology
|September 18, 2023
概括
一个新的CRISPR/Cas9尼克酶 (nCas9) 工具降低了毒性,并提高了Streptomyces基因组编辑中的生存率. 这种方法有效地修改了低DNA转换效率的菌株中的基因,使得天然产品的发现成为可能.
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:
- 菌株对于生产用于医学和农业的天然产品至关重要.
- 克里斯普尔/Cas9基因组编辑促进了NP的过度生产和发现,但表现出宿主毒性,限制了其在低效率转化菌株中的使用.
研究的目的:
- 为Streptomyces开发一种基于CRISPR/Cas9尼克酶 (nCas9) 的低毒性基因组编辑工具.
- 提高Streptomyces菌株的基因组编辑效率和生存率,特别是那些DNA转换效率低的菌株.
主要方法:
- 在Streptomyces coelicolor M145.5.中设计了一个CRISPR/Cas9尼克酶 (nCas9) 系统.
- 与野生类型的Cas9相比,评估的nCas9毒性和细胞存活率.
- 应用了nCas9工具对单个和多个基因/基因集群的删除.
- 在工业性拉巴胺生产菌株Streptomyces rapamycinicus中验证了nCas9工具.
主要成果:
- 与Cas9相比,nCas9显著降低了宿主毒性和提高了细胞存活率.
- 在单基因 (87-100%) 和基因集群 (63-87%) 删除中实现了高效率.
- 在同时删除两个基因或基因集群时,获得了分别为47%和43%的效率.
- 在Streptomyces rapamycinicus中成功应用nCas9进行基因删除,效率为27.2-30%,其中Cas9的效率较低.
结论:
- 克里斯普尔/nCas9系统提供了一种低毒性,高效的Streptomyces基因组编辑方法.
- 这种工具克服了CRISPR/Cas9在DNA转换效率低的菌株中的局限性.
- 促进基因工程以提高天然产品产量和在Streptomyces中发现新的化合物.
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