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相关概念视频

CRISPR01:59

CRISPR

49.1K
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...
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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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CRISPR and crRNAs

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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.
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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相关实验视频

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Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP
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在PAM相邻的DNA灵活性调中,CRISPR-Cas12a的目标外结合.

Aleique Allen1, Brendon H Cooper2,3, Jaideep Singh1

  • 1Department of Chemistry, University of Southern California, 3430 S Vermont Ave., Los Angeles, CA, 90089, USA.

Scientific reports
|February 10, 2025
PubMed
概括

在原体空间邻基因 (PAM) 附近的DNA灵活性会影响Cas12a核酶的目标外结合. 在PAM+1,+2和+3位置的脱对增加了Cas12a的目标外活动,影响了基因组编辑应用程序.

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科学领域:

  • 分子生物学分子生物学
  • 遗传学 是一个遗传学.
  • 生物化学 生物化学

背景情况:

  • Cas12a是一种用于基因组操纵的CRISPR相关核酶.
  • 在Cas12a目标识别中,与原空间邻基因 (PAM) 邻的DNA灵活性是相关的.

研究的目的:

  • 调查PAM相邻DNA灵活性和Cas12a目标外结合之间的关系.
  • 了解PAM附近的DNA序列变异如何影响Cas12a的特异性.

主要方法:

  • 调整了一种SELEX-seq方法来分析PAM+1到+6位置的DNA-DNA不匹配.
  • 使用FnCas12a进行了体外结合测试,使用DNA库进行了不匹配的测试.
  • 结合和不结合的DNA群体的测序确定了非目标结合倾向.

主要成果:

  • 卡斯12a的非目标结合取决于PAM相邻的DNA灵活性.
  • 需要在PAM+1的protospacer脱对,才能实现目标外的结合.
  • 在PAM+2和+3位置脱配时,目标外结合会增加.

结论:

  • 与PAM相邻的DNA灵活性可以调节Cas12a的目标外结合.
  • DNA 的物理性质影响了Cas12a 的目标歧视.
  • 这些发现对优化基于Cas12a的基因组编辑工具有意义.