相关实验视频

Updated: Jul 5, 2025

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Published on: May 10, 2020

克里斯普尔-Cas12a用于高效且无标记物的向集成到人类多能干细胞中

Ruba Hammad^1,2,3,4, Jamal Alzubi^1,2, Manuel Rhiel^1,2

¹Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany.

International journal of molecular sciences

|January 23, 2024

概括

CRISPR-Cas12a Ultra能够在人类细胞中进行高效的基因编辑,达到90%以上的破坏性和高效的无选择性淘汰. 这种强大的基因组编辑工具对干细胞的治疗应用具有前景.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
细胞和基因疗法细胞和基因疗法

背景情况:

克里斯普尔-Cas12a系统是基因组编辑的强大工具,以其分层的DNA双链断裂机制而闻名,其目标是富含AT的PAM序列.
工程 Cas12a 变体的开发,如 Cas12a Ultra,已扩大其适用于各种细胞类型,包括原始人体细胞.

研究的目的:

评估CRISPR-Cas12a Ultra在人类T细胞,造血干细胞和原生细胞 (HSPC) 以及诱导多能干细胞 (iPSC) 内的基因破坏和敲门应用中的效率.
证明CRISPR-Cas12a Ultra在精确基因整合方面的潜力,特别是针对人类iPSC中的AAVS1安全港区和CSF2RA基因.

主要方法:

使用CRISPR-Cas12a超级核糖蛋白复合体,用于人类细胞系和原始细胞中的基因编辑实验.
使用定量方法在多个目标部位评估基因破坏频率.
进行了有针对性的敲门实验,将GFP标记物和CSF2RA超外显子集成到人类iPSC的特定基因组位置,而无需选择.

主要成果:

在人类T细胞,HSPC和iPSC中达到超过90%的等位基因破坏频率.
在人类iPSC中高达90%的等位基因中,在不需要选择的情况下,有效地将GFP基因集成到AAVS1部位,并将CSF2RA超外显子集成到CSF2RA.
在超过50%的选iPSC克隆中确认了双基融合,保持了多能性和基因组完整性.

结论:

CRISPR-Cas12a Ultra是一个高效的基因组编辑平台,用于人类iPSC的基因破坏和向敲击.
将CRISPR-Cas12a Ultra与腺相关病毒载体相结合,为干细胞应用中精确的基因整合提供了一个强大的策略.
这项技术具有很大的潜力,可以在人类细胞中推进治疗基因组工程.

关键词:

在 AAV AAV AAV 中.克里斯普尔是什么意思?克里斯普尔是什么意思?这就是Cas12a.这就是Cpf1pf.在Cpf1 Ultra中使用.在HSC上,HSC是高质量的.在HSPC中,HSPC是最重要的.一个T细胞细胞.基因组编辑基因组编辑这就是 iPSC 的意义.

更多相关视频

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

Published on: November 1, 2024

相关概念视频

Induced Pluripotent Stem Cells

Induced Pluripotent Stem Cells

Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have differentiated into a specific cell type are post-mitotic—that is, they no longer divide. However, scientists have found a way to reprogram these mature cells so that they “de-differentiate” and return to an unspecialized, proliferative state. These cells are also pluripotent like embryonic stem cells—able to produce all cell types—and are therefore...

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...