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可切割的捐赠者辅助CRISPR/Cas9系统显著提高了在Physcomitrium patens中大DNA插入的效率

Xinyuan Gu¹, Jintao Lang¹, Ying Chang¹

¹College of Life Sciences, Capital Normal University, Beijing, 100048, China.

The Plant journal : for cell and molecular biology

|February 21, 2025

概括

我们开发了一种体内可切割 (IVC) 供体系统,以提高CRISPR/Cas9基因组编辑效率,用于中的大型DNA插入. 这种方法显著提高了敲进率,为遗传研究和作物改进提供了强大的工具.

科学领域:

植物生物技术植物生物技术
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:

克里斯普尔/Cas9能够实现精确的基因组编辑,但在很大的DNA插入内敲效率方面存在困难.
较大的外源DNA片段导致基因组编辑应用中的敲入成功率降低.

研究的目的:

开发和评估一个体内可切割 (IVC) 的捐赠者辅助的CRISPR/Cas9系统,用于增强Physcomitrium patens (P. patens) 中的大片段敲击.
为了提高同质导向修复 (HDR) 的效率,在中插入大量的DNA序列.

主要方法:

携带CRISPR/Cas9等离子体和一个含有两个Cas9位点的IVC捐赠体,并排在同质模板的P. patens原生质中.
在目标位置评估了两个sgRNA和四个DNA插件 (5.8,7.5,11.1和7.4kb).
对IVC捐赠者效率与标准循环捐赠者进行敲门率比较.

主要成果:

在PpPDV2-4部位,IVC供体显著提高了CRISPR/Cas9介导的5.8,7.5和11.1kb片段的敲进效率,整合率分别从29.6%提高到67.8%,15.0%提高到72.0%,12.1%提高到65.6%.
在Pp6c18_3160位点使用IVC捐赠体的7.4kb片段敲入也显示出较高的效率与标准的循环捐赠体相比.
IVC捐赠系统有效地促进了HDR的线性模板在体内释放.

结论:

在IVC捐赠者辅助的CRISPR/Cas9系统是一个强大的工具,用于有效的大片段敲击在P. patens.
这一策略有可能用于基础研究,中的合成生物学,以及通过原生质转化改善作物特征.

关键词:

基于CRISPR/Cas9的基因向定位在IVC捐赠体等离子体中.菲斯科米特里姆帕斯 (Physcomitrium patens) 是一个古老的生物.同质性导向的维修.大片段插入插入.技术提前技术提前

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