Cas9-嵌入超活跃的TadA8e赋予了高效和高特异性的A-to-G基编辑在米中
Jianjian Hu1, Xue Li2, Yuhong Gao1
1State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization, Sanya Institute of Nanjing Agricultural University, Jiangsu Engineering Research Center for Plant Genome Editing, National Observation and Research Station of Rice Germplasm Resources, Nanjing Agricultural University, Nanjing, China.
Plant biotechnology journal
|December 23, 2025
概括
工程基编辑器 (ABEs) 减少了非目标基因组编辑. 化学高保真ABEs (CH-ABEs) 提高了精度,并创造了基因编辑大米,包括抗除草剂的耐药性.
科学领域:
- 分子生物学分子生物学
- 基因组编辑 基因组编辑
- 植物科学 植物科学
背景情况:
- 腺基编辑器 (ABEs) 能够在没有双链断裂的情况下实现精确的A-to-G转换.
- 在ABE中过度活跃的腺脱氨酶TadA8e可以导致无意的全基因组非目标编辑.
研究的目的:
- 设计更安全,更具特异性的腺基编辑器 (ABE).
- 在ABE中最大限度地减少sgRNA依赖和sgRNA独立的目标外效应.
- 应用增强的ABE来创建具有理想特征的基因编辑大米.
主要方法:
- 通过将TadA8e嵌入Cas9尼卡酶中,为ABE (CP-ABE) 设计了11种仿制蛋白.
- 使用高保真Cas9变体开发了四个模拟高保真ABEs (CH-ABEs).
- 使用全基因组和全转录组测序 (WGS/WTS) 来确认特异性.
- 整合了Sniper2L和PAM-less SpRY变体,以进一步提高编辑器的性能.
主要成果:
- 四个CP-ABE表现出强大的目标活动,减少了目标之外的编辑.
- 通过CH-ABE实现了非目标编辑的显著减少 (高达7.0倍和79.4倍).
- CH-ABEs产生了22.0%-72.4%的同卵性和双性米种突变.
- 通过引入特定的突变,增强的ABE成功地产生了抗除草剂的耐药大米.
结论:
- 工程 CP-ABE 和 CH-ABE 显著提高了腺基编辑的安全性和特异性.
- 开发的CH-ABE是生成植物精确基因编辑的有效工具,以创造抗除草剂的米为例.
- 使用Sniper2L和SpRY变体的进一步增强扩大了基础编辑器的实用性和准范围.
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