在使用DIPA-CRISPR的极度耐受性伴生遗传迟体中,单步生成同卵性淘汰/淘汰个体

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Updated: Jun 24, 2025

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

Published on: July 12, 2017

Koyuki Kondo^1,2, Akihiro Tanaka¹, Takekazu Kunieda¹

¹Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.

|June 13, 2024

概括

研究人员在迟动物中实现了遗传基因编辑,使得研究它们极端耐压力机制成为可能. 这一突破首次允许同卵性结合体的淘汰和淘汰迟等级个体.

科学领域:

后期生物学的生物学.
基因编辑技术基因编辑技术
极端爱好者的研究是极端爱好者的研究.

背景情况:

晚级动物对极端环境条件表现出了显著的耐受性.
了解性弹性的体内遗传机制至关重要.
以前的体细胞基因编辑并没有产生遗传性变化.

研究的目的:

建立一种方法来生成基因编辑后期变种个体.
为了能够在体内分析晚级弹性机制.
为了促进在Tardigrada的遗传研究.

主要方法:

在Ramazzottius varieornatus中使用了直接的父母CRISPR (DIPA-CRISPR) 方法.
微注射缩的Cas9核糖核蛋白 (RNP) 进入父母的晚年雌性.
同注射的单链寡度氧核酸 (ssODN) 用于敲门实验.

主要成果:

产生了基因编辑的G0晚期后代,主要具有同卵性突变.
使用ssODNs实现了目标基因的同卵性内置.
在随后的G1/G2世代中证明了编辑过的等位基因的遗传性.

结论:

这项研究介绍了第一个成功的遗传基因编辑在类Tardigrada.
开发的方法允许直接生成同卵性淘汰/敲进型迟缓类动物.
这有助于体内研究晚级极端耐受性和进化发育生物学 (Evo-Devo).

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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.