生物发光记者系统的CRISPR/Cas9诱导的恢复用于植物单细胞基因表达分析
Ryohei Ueno1, Shogo Ito1, Tokitaka Oyama2
1Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Scientific reports
|November 21, 2025
概括
我们开发了一种新的CRISPR/Cas9诱导的生物发光记者系统 (CiRBS) 恢复,用于植物基因表达分析. 这种方法使得可靠的长期,单细胞监测基因活动和在Arabidopsis的波动.
科学领域:
- 植物科学 植物科学
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
背景情况:
- 生物发光监测对于理解生物调节系统,包括植物昼夜钟至关重要.
- 目前用于植物单细胞基因表达分析的方法由于影响记者信号的生理因素变异存在局限性.
- 使用 luciferase 记者进行过渡性转染,但可能会受到生理波动的影响.
研究的目的:
- 开发一种新且可靠的CRISPR/Cas9诱导的记者系统,用于长期监测Arabidopsis的单细胞生物发光.
- 通过实现基因组集成的记者基因表达分析来克服现有方法的局限性.
- 为了能够精确量化细胞间异质性和基因表达的时间波动.
主要方法:
- 在转基因Arabidopsis中开发了CRISPR/Cas9诱导的生物发光记者系统 (CiRBS) 恢复.
- 使用了一个不活跃的 luciferase 突变体 (LUC40Ins26bp) 与 26-bp 插入.
- 通过使用CRISPR/Cas9通过粒子轰炸在插入部位引入一个INDEL恢复了酶活性.
- 从植物基因组中集成的记者基因中监测恢复的生物发光.
主要成果:
- 使用CiRBS成功实现了Arabidopsis细胞生物发光的长期监测.
- 在CRISPR/Cas9诱导后的24小时内,英德尔的形成和重组在很大程度上完成了.
- 在7.2%的CRISPR/Cas9移植细胞中恢复了生物发光.
- 估计94%的生物发光恢复的细胞有一个单个,最佳的重组染色体.
结论:
- CiRBS为植物单细胞基因表达分析提供了一种可靠的方法.
- 该系统允许准确评估细胞间异质性和时间基因表达动态.
- CiRBS能够从单个基因组位点进行强大的监测,克服以前的局限性.
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