相关实验视频

Updated: Jul 19, 2025

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Published on: April 10, 2018

用双点击sgRNA-Cas9复合体破坏蛋白质表达:一种对CRISPR基因编辑的模块化方法

Santiago Tijaro-Bulla¹, Eiman A Osman¹, Chris D St Laurent¹

¹Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.

ACS chemical biology

|August 9, 2023

概括

化学修饰的单向导RNA (sgRNA) 片段可以增强CRISPR-Cas9基因编辑. 这种模块化方法改善了核酶耐药性,并使多个基因标的同时破坏成为可能.

科学领域:

分子生物学分子生物学
生物化学生物化学
合成生物学合成生物学

背景情况:

CRISPR-Cas9是一种强大的基因编辑工具,但合成功能单向导RNA (sgRNA) 是具有挑战性的.
一种使用铜催化阿酸循环添加的模块化方法已经开发出来,可以从较小的片段组装sgRNA.
提高sgRNA稳定性对于高效和强大的基因编辑应用至关重要.

研究的目的:

为了提高模块化sgRNAs的稳定性和有效性,用于CRISPR-Cas9基因编辑.
为了研究化学修饰对 sgRNA 对核糖核酸酶耐药性的影响.
为了证明化学稳定 sgRNAs 在多重基因编辑中的实用性.

主要方法:

化学合成的sgRNA片段使用铜催化化酸循环添加组装.
修改后的核酸 (2'-O-Me和酸) 被纳入sgRNA的5'和3'端.
评估了CRISPR-Cas9调解基因淘汰,包括同时淘汰Siglec-3和Siglec-7.

主要成果:

在两端加入三种改性核酸,显著提高了sgRNA的稳定性和对核糖酶的抗性.
化学稳定的sgRNAs在Cas9介导的基因淘汰中表现出更高的效率.
两个目标基因 (Siglec-3和Siglec-7) 的同时淘汰成功实现,使蛋白质水平的破坏成为可能.

结论:

模块化sgRNA组装与化学修饰相结合,为创建稳定基因编辑工具提供了一个多功能平台.
这种方法克服了sgRNA合成的局限性,并提高了CRISPR-Cas9的性能.
开发的方法促进了先进的应用,如多重基因编辑,用于同时破坏蛋白质表达.

更多相关视频

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

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

相关概念视频

CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

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...

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...

CRISPR and crRNAs

CRISPR and crRNAs

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...