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外核酶融合的CRISPR-cas系统增强了针对大豆功能基因组学的定向基因组编辑

Vikas Devkar¹, Kaushik Ghose¹, Leonidas D'Agostino¹

¹Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX, 79409, USA.

BMC plant biology

|October 1, 2025

概括

大豆中的外核酶融合CRISPR/Cas系统可实现更大,更有针对性的DNA删除. 这扩大了基因组编辑能力,用于剖析植物调节元素并创建新的等位基因变异.

科学领域:

植物生物技术植物生物技术
基因组编辑基因组编辑
分子生物学分子生物学

背景情况:

克里斯普尔/卡斯系统提供革命性的植物基因组编辑.
现有的系统偏向于小插入/删除 (indels).
这种局限性阻碍了对调节元件的研究和产生多种基变异的研究.

研究的目的:

在大豆中开发和评估外核酶融合的CRISPR/Cas系统.
为了克服当前CRISPR/Cas技术中小型indel偏差的局限性.
为了实现更大规模的,有针对性的删除,以实现增强的基因组编辑.

主要方法:

Cas9和Cas12a与T5外核酶和TREX2.2的工程融合.
在大豆中的GmWOX5位点评估编辑性能.
使用了Agrobacterium rhizogenes介导的转化和深度安普利康测序.

主要成果:

原生Cas9/Cas12a产生了小的删除 (1-10 bp).
T5-Exo融合产生了中度 (26-50 bp) 和大 (>50 bp) 删除.
TREX2融合增强了小 (11-25 bp) 到中度 (26-50 bp) 的删除.
外核酶融合减少了插入,并偏向向PAM近端区域的删除.

结论:

外核酶融合的CRISPR/Cas系统有效地在大豆中产生更大的删除.
这些系统扩展了CRISPR工具包,以准调控元素和微RNA.
这项技术有助于精确设计植物中具有影响力的等位基变异.

关键词:

大豆, 基因编辑, 外核酶, Cas9, Cas12a, 大量删除.

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