与CRISPR相关的转基因酶用于各种蛋白质细菌的向突变发生
Lidimarie Trujillo Rodríguez1, Adam J Ellington1, Christopher R Reisch1
1Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, United States.
ACS synthetic biology
|June 27, 2023
概括
研究人员开发了一种新的CRISPR关联转移酶 (CAST) 系统,用于细菌中精确的基因组工程. 该系统可实现高效的特异性转子子突变发生,简化遗传研究,加速发现基因型-表型联系.
科学领域:
- 分子生物学分子生物学
- 微生物学 微生物学
- 基因组工程是基因组工程.
背景情况:
- 传统的转子子突变发生提供全基因组的基因破坏,但需要艰苦的突变分离.
- 与CRISPR相关的转体酶 (CAST) 系统可以实现可编程的,特定站点的转体子向.
- 有效的遗传工具对于将不同细菌物种的基因型与表型联系起来至关重要.
研究的目的:
- 描述和演示一种用于细菌基因组工程的新型CRISPR相关转移酶 (CAST) 系统.
- 在多个细菌类中评估CAST系统的效率和实用性.
- 展示该系统对单位,多位和代性突变发生的能力,使用大型DNA插入.
主要方法:
- 采用双等离子体策略,表达来自广域宿主等离子体的CAST基因,并从pUC等离子体中导向RNA/转子体.
- 在CAST系统中测试了单基因干扰,多位点集成和大转子子插入 (超过11kbp) 在蛋白质细菌中.
- 进行了代转体子突变发生,以评估系统的可重复使用性和效率.
主要成果:
- 在Beta和Gammaproteobacteria (例如,Burkholderia thailandensis*,Pseudomonas putida*) 中,单基因破坏效率接近100%.
- 在Alphaproteobacterium *Agrobacterium fabrum*中实现了45%的峰值效率.
- 在两个位点同时进行协同集成,大转子体插入 (>11 kbp) 和代性突变发生被证明具有高效率.
结论:
- 描述的CAST系统为各种细菌的特定基因组工程提供了一个强大而高效的工具.
- 它对单个和多个位置的修改,大有效载荷能力和代使用的能力显著简化了遗传研究.
- 这个系统对于在各种研究领域推进基因组工程具有很大的前景.
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