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相关概念视频

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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...
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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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CRISPR01:59

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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...
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Homologous Recombination02:31

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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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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.
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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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具有提高特异性的理性工程 Cas9 核酶

Ian M Slaymaker1, Linyi Gao2, Bernd Zetsche1

  • 1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Science (New York, N.Y.)
|December 3, 2015
PubMed
概括
此摘要是机器生成的。

被称为增强特异性Cas9 (eSpCas9) 的工程 Cas9 变种显著减少基因组编辑中的意外DNA分裂. 这些改进的Cas9工具保持了更安全的基因编辑应用的精确定位.

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科学领域:

  • 分子生物学
  • 生物技术
  • 基因组学

背景情况:

  • 由RNA引导的内核酶Cas9是基因组编辑的关键工具,使得有针对性的DNA修改成为可能.
  • Cas9酶在特定的基因组位置创建双链断裂 (DSB),由RNA分子指导.
  • Cas9的一个显著的局限性是它可能会分裂目标位置,从而损害编辑精度和安全性.

研究的目的:

  • 来自Streptococcus pyogenes Cas9 (SpCas9) 的增强特异性Cas9 (eSpCas9) 变种的工程.
  • 严格评估eSpCas9变体在人类细胞中的特异性和目标裂变活性.
  • 在基因组编辑应用中解决非目标裂变的挑战.

主要方法:

  • 使用结构指导蛋白质工程来修改SpCas9.
  • 针对性深度测序用于检测潜在的非目标部位的Cas9介导的DNA裂变.
  • 进行了无偏见的全基因组脱分析,以全面评估整个基因组的Cas9活性.

主要成果:

  • 与野生类型的SpCas9相比,工程化eSpCas9变体显示出非目标DNA裂变的显著减少.
  • eSpCas9变种保持了高目标切割效率,确保在所需位置精确编辑.
  • 针对性和全基因组分析都证实了eSpCas9变异的增强特异性.

结论:

  • 增强特异性SpCas9 (eSpCas9) 变种有效地减少基因组编辑中的非目标效应.
  • eSpCas9保留了强大的目标分裂活动,使其成为精确基因编辑的可靠工具.
  • 这些工程 Cas9 变种为需要高特异性的多种基因组编辑应用提供了更好的安全性和有效性.