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相关概念视频

CRISPR and crRNAs02:53

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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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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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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What is Genetic Engineering?00:49

What is Genetic Engineering?

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Overview
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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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相关实验视频

Updated: Jul 5, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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使用CRISPR相关的转基因酶进行细菌基因组工程.

Diego Rivera Gelsinger1,2, Phuc Leo H Vo3,4, Sanne E Klompe1,5

  • 1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

Nature protocols
|January 12, 2024
PubMed
概括
此摘要是机器生成的。

与CRISPR相关的转基因酶使高效的,千基基量级的细菌基因组工程能够在没有同源重组的情况下实现. 本协议详细介绍了使用CRISPR关联转移酶 (CAST) 系统来精确整合不同细菌中的遗传有效载荷.

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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相关实验视频

Last Updated: Jul 5, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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科学领域:

  • 分子生物学分子生物学
  • 基因组学就是基因组学.
  • 合成生物学 合成生物学

背景情况:

  • 与CRISPR相关的转基因酶 (CAST) 为大规模基因组工程提供了一个新的平台.
  • 现有的方法通常需要同源重组,并与大量的遗传有效载荷作斗争.
  • CAST系统提供精确的,可编程的DNA序列集成.

研究的目的:

  • 通过CAST系统提出细菌基因组工程的详细协议.
  • 为矢量选择提供指导方针,指导RNA和DNA有效负载定制和传递方法.
  • 引入用于指导RNA设计和多重复合DNA插入的计算工具.

主要方法:

  • 利用CRISPRRNA引导的转基因酶在大肠杆菌中进行基因组插入.
  • 开发了一种用于设计指导RNA的计算算法,以最大限度地减少非目标效应.
  • 实施了CRISPR数组克隆管道,用于多重复合DNA插入.

主要成果:

  • 在大肠杆菌中实现了近100%的基因组插入效率.
  • 在各种格拉姆阴性细菌中证明了CAST系统的强大功能.
  • 通过使用多个指南编程启用多重编辑.
  • 在1-2周内通过新的基因组整合来促进克隆菌株的隔离.

结论:

  • 与CRISPR相关的转基因酶系统代表了千基基基因基因工程的强大工具.
  • 提出的协议简化并提高了细菌基因组修改的效率.
  • 这项技术在设计各种细菌物种方面具有广泛的适用性.