F. rodentium Cas9 的结构和功能基础为CRISPR-Cas蛋白质工程提供了洞察力
Mei Yang1, Siqi Liu2, Guanqiao Chen3
1Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong, China; Molecular Imaging Center, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong, China; Guangdong-Hong Kong-Macao University Joint Laboratory of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong, China.
Cell genomics
|October 17, 2025
概括
在Faecalibaculum动物Cas9 (FrCas9) 系统提供卓越的基因编辑精度. 结构和突变研究揭示了提高效率和减少目标外影响的机制,为先进的CRISPR技术铺平了道路.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 遗传学 是一个遗传学.
背景情况:
- 类类动物的Cas9 (FrCas9) 系统表现出比SpCas9更强大的基因编辑能力,特别是针对像TATA盒这样的真核突变促进体区域.
- 在FrCas9的高精度和效率的基础上,精确的分子机制,特别是其独特的原始空间器相邻动机 (PAM) 识别,以前没有被描述.
研究的目的:
- 阐明FrCas9CRISPR-Cas9系统增强基因编辑精度和效率的结构和分子基础.
- 研究特定结构元素 (如酸锁环) 在调节 FrCas9 的活性和特异性的作用.
主要方法:
- 使用冷电子显微镜 (cryo-EM) 来确定FrCas9单导向RNA (sgRNA) -DNA复合体在关键功能状态 (R循环扩张和前催化) 中的结构.
- 对关键的FrCas9残留物进行了广泛的突变分析,包括酸锁环和PAM遥远区域的残留物,以评估它们对基因编辑活动和特异性的影响.
- 生物化学测试可能用于量化编辑效率和非目标效应.
主要成果:
- 冷-EM结构揭示了FrCas9对5'-NRTA-3'PAM的独特识别,以及对sgRNA-DNA异重复的不寻常超.
- 鉴定出酸锁环对于微调FrCas9的非目标灵敏度和催化效率至关重要.
- 酸锁循环和PAM远程区域的特定替换协同提高了编辑精度和效率.
结论:
- 这项研究为FrCas9CRISPR-Cas9系统提供了前所未有的结构洞察力,解释了其增强的性能.
- 酸锁环和PAM远端区域是FrCas9准确性和功效的关键决定因素.
- 这些发现为设计下一代CRISPR技术提供了一个分子框架,其特异性和效率得到了提高.
相关概念视频
CRISPR
57.5K
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...
57.5K
CRISPR/Cas9 Genome Editing
1.7K
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...
1.7K
CRISPR and crRNAs
18.7K
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...
18.7K
Homologous Recombination
62.6K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
62.6K
The Antiviral System of Bacteria and Archaea: CRISPR
617
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...
617
Conservative Site-specific Recombination and Phase Variation
6.6K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.6K


