克里斯普尔-Cas9淘汰屏幕报告了对Komagataella phaffii分泌基因组的有效减少
Neil C Dalvie1,2, Timothy R Lorgeree2, Yuchen Yang1,2
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Microbial cell factories
|July 31, 2024
概括
在Komagataella phaffii酵母中删除非必需基因显著增加了重组蛋白质的生产. 这种基因工程方法提高了诸如人血清白蛋白和单克隆抗体等有价值蛋白的产量.
科学领域:
- 微生物学 微生物学
- 生物技术是生物技术.
- 合成生物学 合成生物学
背景情况:
- 科马格塔拉法菲是重组蛋白制造的关键酵母.
- 通过基因工程改善分泌的蛋白质标位至关重要.
- 有限的基因注释和基本性知识阻碍了K. phaffii的基因工程.
研究的目的:
- 在K. phaffii. 中,将细胞资源从内源转向重组蛋白质生产.
- 通过识别和破坏非必要的内源基因来克服基因工程的局限性.
- 为了增强K. phaffii. 的重组蛋白质的产生.
主要方法:
- 使用质谱和信号预测识别了内源性分泌蛋白质.
- 开发了一个聚合的指导RNA库,用于CRISPR-Cas9介导的秘密基因的淘汰.
- 预测基因基本性以指导代基因破坏.
- 通过删除多达11个非必不可少的基因来改造K. phaffii菌株.
主要成果:
- 实现了人血清白蛋白产量的约20倍增加.
- 显示单克隆抗体生产增加了两倍.
- 显示破坏仅6个基因可以显著提高重组蛋白产量.
结论:
- 破坏K. phaffii中的特定内源基因有效地增加了重组蛋白质的生产.
- 进一步减少内源性蛋白质组有可能提高菌株的性能.
- 开发的CRISPR-Cas9指南库和基本性数据将有助于K. phaffii在未来对各种重组蛋白和酶的工程工作.
相关概念视频
CRISPR
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR and crRNAs
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
CRISPR/Cas9 Genome Editing
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...


