CROPseq-multi:用于在聚合的CRISPR屏幕中进行多重扰动和解码的多功能解决方案.
Russell T Walton1,2, Yue Qin1,3, Paul C Blainey1,2,4
1Broad Institute of MIT and Harvard, Cambridge, MA, USA.
bioRxiv : the preprint server for biology
|April 1, 2024
概括
研究人员开发了CROPseq-multi,这是使用Streptococcus pyogenes (Sp) Cas9.9进行多重遗传查的新型晶状病毒系统. 该工具有效地将基因扰动与mRNA条形码结合起来,用于先进的聚合查应用.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 通过分析基因扰动和由此产生的表型,向前遗传选对于理解生物系统至关重要.
- 多重化遗传干扰提高了查效率,并使复杂的遗传相互作用的研究成为可能.
- 现有的多重复合工具经常与需要嵌入mRNA条形码的聚合选方法发生冲突,这限制了它们在单细胞测序等技术中的使用.
研究的目的:
- 开发一种新型的lentiviral系统,CROPseq-multi,用于多重化Streptococcus pyogenes (Sp) Cas9基扰动与mRNA嵌入条形码.
- 确保与聚合查方法的兼容性,包括使用单细胞测序的方法.
- 提高多重基因查的效率和适用性.
主要方法:
- 在CROPseq的启发下,开发了一个lentiviral系统 (CROPseq-multi).
- 整合Streptococcus pyogenes (Sp) Cas9用于遗传干扰.
- 纳入mRNA嵌入条形码,以实现聚合查兼容性.
- 对光学聚合屏幕的现场检测协议的优化.
主要成果:
- CROPseq-multi表现出相当于CROPseq的每指导活性,具有较低的lentiviral重组.
- 该系统与缩选和光学聚合屏幕兼容.
- 优化的协议提高了光学聚合屏幕的条形码检测效率10倍和解码效率3倍.
- CROPseq-multi可以扩展到单细胞测序读数.
结论:
- CROPseq-multi提供了一种使用SpCas9.9进行多重基因查的多功能解决方案.
- 这一进步扩大了聚合查方法的适用性,特别是那些需要嵌入mRNA的条形码的方法.
- 促进对遗传相互作用和复杂的生物系统进行更深入的研究.
相关概念视频
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...
02:46Genetic Screens
Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes...
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes...
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...
01:59CRISPR
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...