相关实验视频
Updated: Sep 22, 2025

11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
27.6K
通过IscB-ωRNA引导DNA裂变的结构基础和与Cas9的机制比较
Gabriel Schuler1, Chunyi Hu1, Ailong Ke1
1Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.
概括
像Cas9这样的2类CRISPR受体可能是从转子核酶进化而来的. IscB-ωRNA结构与Cas9有相似之处,解释了DNA裂变机制和ωRNA
科学领域:
- 分子生物学
- 结构生物学
- 遗传学
背景情况:
- 包括Cas9和Cas12在内的CRISPR-Cas系统是强大的基因编辑工具.
- 这些系统被假设是从移动基因元素如IS200/IS605转位子进化而来的.
- IscB是一个较小的Class 2效应器,具有与Cas9相似的域组织,利用 ωRNA进行DNA向和分裂.
研究的目的:
- 阐明与双链DNA (dsDNA) 结的IscB-ωRNA复合物的高分辨率结构.
- 揭示IscB和Cas9核糖蛋白之间的机制和结构相似之处.
- 了解 ωRNA 和特定域在 IscB 中介的 DNA 裂变中的作用.
主要方法:
- 使用冷电子显微镜 (cryo-EM) 来确定IscB-ωRNA-dsDNA复合物的结构.
- 进行了高分辨率结构分析以可视化分子相互作用.
- 使用突变分析来评估IscB特定PLMP域的可用性.
主要成果:
- 一个2.78-angstrom的冷-EM结构揭示了IscB-ωRNA-dsDNA复合体内的详细相互作用.
- 关键的机制步骤,包括目标相邻的模式识别,R循环形成和dsDNA裂变,在高分辨率下可视化.
- ωRNA 的功能与 Cas9 的 REC 域类似,与 RNA-DNA 异质复合体相互作用,而 PLMP 域被发现是不可用的.
结论:
- 这项研究为IscB的结构和功能提供了原子层面的见解,强调了它与Cas9的进化关系.
- 这些发现解释了IscB如何利用 ωRNA引导dDNA分裂,反映了Cas9的功能方面.
- 从祖先的 IscB 转变为 Cas9 可能涉及显著的变化,包括 ωRNA 减少和蛋白质域演变.
相关概念视频
CRISPR
53.1K
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...
53.1K
CRISPR and crRNAs
17.5K
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...
17.5K
CRISPR/Cas9 Genome Editing
443
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...
443
Ribozymes
12.5K
The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can...
Ribozymes can...
12.5K
RNA Interference
26.5K
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
26.5K
Single-Strand DNA Binding Proteins
15.1K
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
15.1K

