阶段:基于TnpB的紧且多功能基因组编辑工具包
Jing Luo1, Natalie Chia1, Yuxi Qin1
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
概括
一个名为STAGE的新工具包能够精确地编辑Streptomyces细菌的基因组. 这种基于TnpB技术的系统为合成生物学应用提供了高效的基因编辑和基因编辑.
科学领域:
- 微生物学
- 分子生物学
- 生物技术
背景情况:
- 链杆菌对于生产天然产品具有价值,并且由于众多未表征的生物合成基因集群,它们是合成生物学的目标.
- 现有的CRISPR-Cas系统对Streptomyces有局限性,包括细胞毒性和大效应蛋白大小,阻碍了更广泛的应用.
研究的目的:
- 开发一种新,高效,多功能的Streptomyces基因组编辑平台.
- 在Streptomyces操纵中克服当前遗传工具的局限性.
主要方法:
- 使用小型ISDra2 TnpB效应器开发与Streptomyces兼容的TnpB辅助基因组编辑 (STAGE) 工具包.
- 实施STAGE用于特定位点的基因突变,并开发STAGE-cBEST/STAGE-McBEST用于C·G·T·A基编辑.
- 通过人工智能辅助的蛋白质工程来提高ISDra2 TnpB的效率,并识别一个功能性的TnpB正态.
主要成果:
- 在工业性Streptomyces菌株中,STAGE展示了高效的,特定位点的基因编辑,具有最小的非目标效应.
- STAGE-cBEST和STAGE-McBEST在单个和多重编辑基础上实现了超过75%的效率.
- 工程TnpB变种达到近100%的基因组编辑效率,并且TnpB的正确性也得到了验证.
结论:
- STAGE是一个精确,可编程和多功能的Streptomyces基因组编辑平台.
- 这套工具可以促进工业相关的Streptomyces物种的先进基因操纵和合成生物学.
- 像STAGE这样更小,更高效的编辑工具的开发显著提升了Streptomyces在生物技术中的潜力.
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