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相关概念视频

CRISPR01:59

CRISPR

50.8K
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...
50.8K
CRISPR and crRNAs02:53

CRISPR and crRNAs

17.0K
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...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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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...
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Homologous Recombination02:31

Homologous Recombination

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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...
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相关实验视频

Updated: Jun 29, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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细胞驱动的Cas9核酶的定向进化.

Giulia Vittoria Ruta1, Matteo Ciciani2,3, Eyemen Kheir2

  • 1Laboratory of Molecular Virology, Department CIBIO, University of Trento, Trento, Italy. giuliavittoria.ruta@unitn.it.

Genome biology
|March 26, 2024
PubMed
概括

科学家们开发了一个真核生物平台来改善病例活动 (EPICA),以增强基因组编辑工具. 这个平台成功地改善了Campylobacter jejuni Cas9 (CjCas9) 核酶,在哺乳动物细胞中产生了具有显著更高活性和特异性的UltraCjCas9.

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科学领域:

  • 分子生物学分子生物学
  • 生物技术是生物技术.
  • 基因编辑技术的技术

背景情况:

  • 基因组编辑的进步需要具有更好的真核细胞兼容性的工具.
  • 许多Cas9变种显示出潜力,但需要优化以实现高效的基因组编辑.
  • 坎皮洛巴克特 (Campylobacter jejuni) Cas9 (CjCas9) 的小尺寸有利于在哺乳动物细胞中传递,但其编辑活动有限.

研究的目的:

  • 开发一种定向进化平台,以增强弱活性Cas9核酶的活性.
  • 为了创建一个更活跃和特定的CjCas9变体用于真核生物基因组编辑应用程序.

主要方法:

  • 使用酵母辅助细胞选择和哺乳动物细胞记者系统开发了改善细胞活动的真核细胞平台 (EPICA).
  • 员工指导进化通过代选择和选来增强CjCas9活动.
  • 在哺乳动物内源性基因组位点中验证了增强的Cas9变体.

主要成果:

  • 在EPICA平台上成功生成了一种增强的CjCas9变种,命名为UltraCjCas9.
  • 与野生型CjCas9相比,UltraCjCas9在哺乳动物细胞中的编辑活性高达12倍.
  • 增强型变种UltraCjCas9在整个基因组中保持高特异性.

结论:

  • 一个新的真核细胞管道 (EPICA) 已建立,用于增强Cas9核酶活性.
  • 这一平台有助于优化用于基因组编辑的自然存在的RNA导向核酶.
  • 超CjCas9的开发表明了EPICA在释放新的基因组编辑工具方面的潜力.