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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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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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CRISPR01:59

CRISPR

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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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DNA-only Transposons02:57

DNA-only Transposons

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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...
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CRISPR and crRNAs02:53

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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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相关实验视频

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用CRISPR关联的转子子用于可编程的病毒载体工程和原始编辑.

Quyen T Dang1, Chin-Wei Chang1, Pin-Yan Chen1

  • 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300044, Taiwan.

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概括

SHOT 2.0 是一种新的CRISPR相关的转子子平台,用于E. coli中的灵活的细菌工程. 这种工具可以在各种细胞类型中实现高效的基因传递和原始编辑,从而扩大了baculovirus的应用.

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科学领域:

  • 分子生物学分子生物学
  • 基因编辑 基因编辑
  • 病毒学 病毒学

背景情况:

  • 百科病毒被广泛用于重组蛋白质表达和基因传递.
  • 目前的 bacmid 工程方法缺乏灵活性和可编程性.
  • 与CRISPR相关的转子子系统为先进的基因操纵提供了潜在的潜力.

研究的目的:

  • 开发一个优化的CRISPR相关的转子子平台,用于灵活和可编程的细菌体工程.
  • 为了使大型DNA货物能够有效地集成到特定的细菌体位置.
  • 展示该平台用于构建用于基因编辑的先进的baculovirus载体的实用性.

主要方法:

  • 开发SHOT 2.0,一种与CRISPR相关的转体子系统,用于在大肠杆菌中进行RNA引导的巴克米德编辑.
  • 大型DNA有效载荷 (≥14kb) 的特定站点集成到bacmid位点 (v-cath,ODVe56).
  • 构建和测试一个编码主要编辑器 (PE5max) 的全集baculovirus.
  • 在HEK293T细胞,诱导多能干细胞 (iPSCs) 和肝癌细胞中评估主要编辑效率.

主要成果:

  • 通过SHOT 2.0,可以将大型DNA货物 (≥14 kb) 具体集成到已定义的细菌体位置.
  • 在ODVe56位点的整合显著提高了在连续病毒传递期间的转基因稳定性.
  • 该系统兼容Bac-to-Bac®工作流,允许双基因插入.
  • 矢量介导的原始编辑在HEK293T细胞中实现了高达85.6%的效率,在iPSC和肝癌细胞中达到37.1%.

结论:

  • SHOT 2.0 显著扩展了 baculovirus 工程工具箱,为基因组编辑提供了一个灵活的平台.
  • 该平台可为各种应用,包括基因传递,创建先进的细菌病毒载体.
  • 在各种细胞类型中表现出强大的主要编辑能力,包括像iPSC和癌细胞这样具有挑战性的细胞.