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Updated: May 8, 2026

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

在Arabidopsis thaliana中分析Cas9-和Cas12a诱导的突变发生

Sylvia de Pater¹, Lycka Kamoen¹, Robin van Schendel²

¹Department of Plant Sciences, Institute of Biology, Leiden University, Leiden, BE, 2333, the Netherlands.

The Plant journal : for cell and molecular biology

|July 25, 2024

概括

使用Cas9和Cas12a核酶的CRISPR基因编辑显示了相似的突变率和T-DNA集成,尽管DNA断裂类型不同. 在植物基因组工程中,Cas12a和Cas9同样有效.

科学领域:

植物遗传学植物遗传学
分子生物学分子生物学
基因组工程是基因组工程.

背景情况:

像Cas9和Cas12a这样的CRISPR技术对于基因改造至关重要.
了解双链断裂 (DSB) 后的DNA修复机制是提高CRISPR精度的关键.
卡斯9和卡斯12a在PAM识别,DSB位置和DNA末端配置上有所不同 (粗与分层).

研究的目的:

量化比较Arabidopsis thaliana中的LbCas12a和Cas9的修复概况.
调查DSB终端配置是否影响修复路径选择.
评估 Cas12a 和 Cas9 介导的 T-DNA 集成的效率.

主要方法:

在Arabidopsis thaliana中LbCas12a诱导的DSB的详细修复概况.
量化比较与之前报告的Cas9诱导的DSB修复配置文件.
长读测序分析修复结果和DNA损失.

主要成果:

非同类末端结合 (NHEJ) 占突变的70%,聚合酶甲基介导末端结合 (TMEJ) 在两个核酶中占30%.
像Cas9一样,Cas12a有效地刺激T-DNA集成,用于基因组工程.
TMEJ导致显著的DNA损失,而Cas9诱导的结DSB显示1bp插入取决于聚合酶兰巴达,与Cas12a的分层DSB不同.

结论:

DSB终端配置并不决定NHEJ和TMEJ之间的修复路径选择.
Cas9和Cas12a在基因组工程应用中表现出相似的疗效.
核酶选择可以灵活,基于PAM序列的可用性.

关键词:

阿拉比多普西斯塔利亚纳.这就是Cas12a.这就是Cas9的情况.在DNA聚合酶兰巴达.双链断裂是双链断裂的原因之一.非同类的末端连接 (NHEJ)聚合酶甲基介导末端结合 (TMEJ)

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相关概念视频

In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

In vitro Mutagenesis

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.