在Corynebacterium glutamicum和Bacillus subtilis中进行RNA引导的DNA转换
Siqi Yang1,2, Jiao Zhu1,2, Xiaojie Zhou1,2
1Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
ACS synthetic biology
|June 22, 2023
概括
与CRISPR相关的转基因酶 (CAST) 能够在像Bacillus subtilis和Corynebacterium glutamicum这样的细菌中进行多重基因组编辑. 优化条件显著提高了插入效率,证明了CAST.
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:
- 与CRISPR相关的转基因酶 (CAST) 是多重基因组编辑的新兴工具.
- 目前CAST的应用仅限于一小部分细菌物种.
- 来自Vibrio cholerae的I-F型CAST为更广泛的细菌基因组工程提供了潜力.
研究的目的:
- 评估Vibrio cholerae型I-F CAST在Bacillus subtilis和Corynebacterium glutamicum中的RNA导向转移的疗效.
- 通过环境和基因修改,优化CAST介导的转化效率.
- 为了证明CAST在工业相关细菌中大型DNA片段集成的潜力.
主要方法:
- 在B. subtilis和C. glutamicum中使用I-F型CAST系统进行了转换试验.
- 调节培养温度以评估其对插入效率的影响.
- 积极选择策略被用来促进大型DNA片段 (高达9kb) 的整合.
主要成果:
- 类型I-F CAST成功诱导了B. subtilis (0.00018%的效率) 和C. glutamicum (0.027%的效率) 的RNA导向转化.
- 将培养温度降至16°C显著提高了B. subtilis的插入效率,达到3.64%.
- 在C. glutamicum中,在货物DNA中结合spectinomycin耐药性使得9 kb的碎片集成效率达到13.4%.
结论:
- 类型I-F CAST系统是有效的基因组编辑在Firmicutes,包括B. subtilis和C. glutamicum.
- 优化的培养条件和选择策略大大提高了CAST的效率和货运能力.
- CAST技术对跨越多种细菌物种的多功能多重基因组编辑具有重大前景.
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