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使用CRISPR-Cas对内源性Chlamydomonas reinhardtii基因进行高效的精确编辑

Adrian Pascal Nievergelt¹, Dennis Ray Diener¹, Aliona Bogdanova¹

¹Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany.

Cell reports methods

|September 6, 2023

概括

我们开发了一种高效的CRISPR-Cas方法,用于Clamydomonas reinhardtii的无痕基因敲定,克服了以前的困难. 这种技术可以实现精确的基因修饰和高通量突变查,而无需可选择的标记物.

科学领域:

* 分子生物学 * 分子生物学
* 遗传学遗传学是一门学科
* 藻类研究

背景情况:

*CRISPR-Cas基因组工程在*Chlamydomonas reinhardtii*中主要集中在基因破坏上.
* 在这个模型生物体中,无痕的诺基因转基因是具有挑战性的.
*以前的方法通常需要先前存在的突变系或产生残留标记.

研究的目的:

* 建立一种高效的同质性导向方法,用于在Chlamydomonas reinhardtii*中进行无痕诺基因突变发生.
* 开发用于生成突变的高通量选和交叉方法.
*通过共同向的插入,促进无标记突变系的产生.

主要方法:

*通过电穿孔传递CRISPR-Cas核核蛋白和线性双链DNA (dsDNA) 供体.
* 开发高通量变压器交叉技术.
* 实施基于定制定量PCR (qPCR) 的查,用于突变物和介质后代.

主要成果:

* 证明了融合标签和序列修改的高效无痕集成.
* 通过共同向插入成功生成了无标记物突变系.
* 确定并提供了在插入卡塞特集成过程中减轻错误突变的策略.

结论:

* 开发的基于CRISPR-Cas的管道显著提高了克拉米多马纳斯强化菌的敲击能力.
*这种方法促进了精确的基因工程,并加速了突变基因的产生.
*这项研究为这种藻类模型的高通量遗传分析提供了强大的工具.

关键词:

克里斯普尔-Cas9是什么意思克拉米多莫纳斯病毒是什么?内源性标记是一种内源性标记.基因组编辑基因组编辑高通量选的高通量选植物生物技术植物生物技术

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