相关实验视频

Updated: Jun 5, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

使用CRISPR-Cas9核酶和捐赠者DNA结构的组合性病毒载体传递的精确基因组编辑

Zhen Li¹, Xiaoling Wang¹, Josephine M Janssen¹

¹Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.

Nucleic acids research

|December 10, 2024

概括

腺相关病毒 (AAV) 和腺病毒 (AdV) 载体有效地提供CRISPR-Cas9基因编辑工具. 将单链AAV与第三代AdV结合起来,可以精确地将整个转基因插入细胞中.

科学领域:

分子生物学分子生物学
基因治疗基因治疗
生物技术是生物技术.

背景情况:

精确的基因组编辑需要有效地传递核酶和捐赠者DNA.
腺病毒 (AdV) 和腺相关病毒 (AAV) 载体正在探索提供基因编辑组件.
克里斯普尔-Cas9系统能够进行有针对性的DNA修改.

研究的目的:

评估AdV和AAV载体,以共同提供CRISPR-Cas9核酶和捐赠者DNA结构.
为了优化基因组编辑效率和精度,使用不同的病毒载体组合.
研究病毒载体设计对基因编辑结果的影响.

主要方法:

使用单链和自我补充的双链AAVs进行捐赠DNA输送.
在CRISPR-Cas9核酶输送中使用第二代和第三代AdVs.
评估基因组编辑的效率,精度和非目标效应.

主要成果:

同时提供单链AAV (HR捐赠者) 和第三代AdV (CRISPR-Cas9) 实现了高达93%的精确整体转基因插入.
克里斯普尔-卡斯9诱导的DNA断裂提高了AAV转导效率.
高保真CRISPR-Cas9核酶减少了有缺陷的AAV基因组的非目标整合.

结论:

AAV和AdV载体为协调交付基因编辑组件提供了一个有效的系统.
这种联合载体方法使得无选择,精确的转基因插入成为可能.
优化的载体选择和核酶忠实性对于安全高效的基因组编辑至关重要.

更多相关视频

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Published on: January 7, 2019

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

相关概念视频

CRISPR

CRISPR

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...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

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...