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从未发芽的阿拉比多普西斯种子在6个A类A类2C蛋白酸酶基因中具有LbCas12a诱导的突变

Cuiping Xin¹, Yu Lu¹, Syeda Leeda Gul¹

¹State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing 100193, China.

Plant physiology

|July 17, 2025

概括

我们开发了最有效的CRISPR/Cas工具,用于Arabidopsis的多重基因组编辑,通过突变六个基因来创建非发芽的种子. 这种Cas12a变体显示了最小的目标偏差和高的编辑效率.

科学领域:

分子生物学分子生物学
植物科学植物科学
基因组编辑基因组编辑

背景情况:

Cas12核酶 (Cas12a,Cas12i,Cas12f) 是多功能基因组编辑工具.
在Arabidopsis thaliana中,各种Cas12变异的多重基因组编辑潜力尚不清楚.

研究的目的:

系统地评估额外的Cas12变体用于Arabidopsis.多重基因组编辑.
为了确定最有效的Cas12变体,并了解影响编辑效率的因素.

主要方法:

使用各种Cas12变体 (LbCas12a,Cas12i3,AsCas12f) 对18个额外目标的系统评估.
对7种LbCas12a变异进行比较分析,重点关注核定位序列和特定突变 (D156R,E795L).
产生T1同卵性六重突变体和评估种子发芽表型.

主要成果:

具有D156R和E795L突变的LbCas12a变体显示出最小的目标偏差和高的编辑效率 (≥73.8%).
超过一半的产生的六重突变体表现出完全的种子发芽停止.
对核定位序列的优化对效率至关重要,E795L突变显示出协同效应.

结论:

开发了最有效的聚类正规间隔短平行体重复 (CRISPR) /CRISPR相关核酶 (Cas) 工具,用于Arabidopsis多重基因组编辑.
通过有效突变六个特定基因,成功生成了非发芽的种子.
Cas12i3在Arabidopsis中也显示出高效率,尽管目标偏差需要进一步细化.

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