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一个优化的CRISPR Cas9和Cas12a突变发生工具包用于大麦和小麦

Tom Lawrenson¹, Martha Clarke², Rachel Kirby²

¹John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK. tom.lawrenson@jic.ac.uk.

|August 13, 2024

概括

这项研究优化了大麦和小麦的CRISPR-Cas9和CRISPR-Cas12a基因编辑系统,实现了单基因和多基因编辑的高效率. 开发的工具箱促进了谷物作物的先进基因组编辑.

科学领域:

植物分子生物学植物分子生物学
基因组编辑技术的技术.
提高作物质量提高作物质量

背景情况:

CRISPR-Cas9和CRISPR-Cas12a是植物基因组编辑的关键可编程核酶.
目前的CRISPR组件存在不同的效率和物种适用性.
优化这些系统对于推进谷物作物研究至关重要.

研究的目的:

为大麦和小麦开发和优化高效的CRISPR-Cas9和CRISPR-Cas12a系统.
创建一个用户友好的工具箱,用于谷物中简单和多重基因组编辑.
为了提高单植物中CRISPR核酶的有效性.

主要方法:

利用子优化的Cas9和Cas12a序列,具有不同的内子数.
用于指导RNA表达的U6和U3促进剂.
实现基于tRNA的多导向数组,用于多重编辑.
集成的GRF-GIF转化促进小麦的卡塞特.

主要成果:

一个Zea mays编码子优化的Cas9与13个内子实现了100%的同时编辑三个目标基因在大麦.
超过90%的小麦T0植物使用优化的Cas9系统对所有三个亚基因组目标进行了编辑.
一个具有8个内子和tRNA多导体阵列的Arabidopsis编码子优化的Cas9产生了大麦中90%的突变等位基因.
在小麦中,Cas12a系统在两个同时向的基因中实现了86%和93%的突变率.
证明多个内核和特定突变 (D156R) 显著提高了Cas12a突变发生效率.

结论:

一个金门模块化克隆系统用于大麦和小麦的Cas9和Cas12a已经开发和提供.
该工具包提供了经过验证的Cas核酶和指导表达选项,用于高效的简单和多重突变发生.
将GRF-GIF录音带纳入小麦可以最大限度地提高基因组编辑工作流程的效率.

关键词:

谷物谷物是一种谷物.基因组编辑基因组编辑黄金大门是黄金大门的一个门.通过内部介导的增强作用.有针对性的突变发生.

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