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闪光基因组编辑管道:一种高效和高通量方法,用于构建数组化的CRISPR库,用于植物功能基因组学

Lu Yao¹, Xiaochun Wang^1,2, Runnan Ke¹

¹National Key Laboratory of Crop Genetic Improvement and Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, People's Republic of China.

Current protocols

|September 27, 2023

概括

我们开发了FLASH基因组编辑管道,用于在植物中进行高通量CRISPR选. 这种方法使用独特的FLASH标签将基因目标与Cas9/gRNA载体联系起来,从而实现高效的基因功能发现.

科学领域:

植物基因组学植物基因组学
基因组编辑技术的技术
农作物育种农作物育种

背景情况:

CRISPR/Cas9是各种生物体中基因编辑的强大工具.
使用CRISPR/Cas9的高通量基因查在植物中具有挑战性.
需要有效的方法来进行植物功能基因组学和作物改进.

研究的目的:

建立一个新的管道,用于在植物中构建阵列的CRISPR库.
为了实现高通量遗传查和在植物中发现基因功能.
开发一种方法,使用独特标识符将目标基因与Cas9/gRNA载体联系起来.

主要方法:

开发FLASH基因组编辑管道,使用12个不同的PCR片段 (FLASH标签) 来索引Cas9/gRNA载体.
组装FLASH标记索引的Cas9/gRNA等离子体,并准备一个等离子体库.
米植物的农细菌媒介转化,使用索引菌株的混合物.
使用常规PCR和凝电泳来分配基因编辑植物中的gRNA信息.

主要成果:

在植物中使用FLASH基因组编辑管道成功构建了一个数组化的CRISPR库.
建立了目标基因/gRNA和FLASH标签之间的独特联系,以有效地检索信息.
证明了在细菌菌株和基因编辑植物中读取gRNA信息的能力.
该协议允许在植物中快速和全面地发现基因功能.

结论:

FLASH基因组编辑管道促进了植物的高通量遗传查.
这种方法可以有效地发现基因功能,并支持作物育种的进步.
该方法为在植物系统中构建和分析数组CRISPR库提供了强大的解决方案.

关键词:

这就是CRISPR/Cas9的作用.排列式图书馆排列式图书馆基因组编辑基因组编辑高吞吐量,具有高吞吐量.植物植物植物植物植物.

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