设计,性能,处理和验证一个聚合的CRISPR扰动屏幕的细菌毒素的设计,性能,处理和验证
Songhai Tian1, Yuhang Qin2, Yuxuan Wu2
1State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China. songhai.tian@pku.edu.cn.
Nature protocols
|November 2, 2024
概括
这项研究提出了一个全基因组聚合的CRISPR选协议,以确定参与细菌毒素通路的宿主因素. 该方法有助于通过分析遗传干扰和细胞反应来了解毒素如何引起疾病.
科学领域:
- 基因组学就是基因组学.
- 分子生物学分子生物学
- 微生物学 微生物学
背景情况:
- 使用聚合集群定期间隔短平行体重复 (CRISPR) 扰动的前进遗传选对于功能基因组学至关重要.
- 克里斯普尔屏幕研究基因型-表型关系,使得研究细胞对细菌毒素等生物药物的反应成为可能.
- 由于各种毒素的多样化作用,研究毒素机制具有挑战性.
研究的目的:
- 为专门设计用于研究细菌毒素的全基因组聚合CRISPR扰动屏幕提供详细的协议.
- 为有效的毒素相关遗传查概述技术考虑,实验步骤和分析方法.
- 为了促进对细胞中毒途径至关重要的宿主因子的发现.
主要方法:
- 全基因组聚合的CRISPR扰动屏幕协议.
- 在细胞群中引入功能增益或功能丧失突变.
- 对扰乱相关的基因型的分析和对特定表型的查.
- 详细步骤涵盖图书馆构建,屏幕执行和结果验证.
主要成果:
- 该协议允许研究细菌毒素对宿主细胞的影响.
- 识别宿主因子,如受体,贩运/转移因子和涉及中毒的基质.
- 适用于广泛的细菌毒素.
结论:
- 开发的CRISPR查协议是剖析细菌毒素与宿主相互作用的强大工具.
- 预计它将揭示关键的宿主因素,调解细胞对毒素的反应.
- 该协议是可适应和可访问的,只需要基本的生物学知识.
相关概念视频
01:59CRISPR
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...
02:53CRISPR 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...
01:59CRISPR
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...
01:23The Antiviral System of Bacteria and Archaea: CRISPR
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this defense.
01:28CRISPR/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...