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扩大CRISPR/Cas9中介基因组编辑的准范围,通过Cas9变异在布拉西卡群中进行编辑

Wenjing Li^1,2, Xuan Li^1,2, Chunyang Wang¹

¹State Key Laboratory of North China Crop Improvement and Regulation, College of Life Sciences, Hebei Agricultural University, Baoding, 071001 China.

|July 8, 2024

概括

工程CRISPR/Cas9变体 (Cas9-NG,SpG,SpRY) 通过准非正规的PAMs,可以在Brassica蔬菜中进行高效的基因组编辑. 这扩大了用于植物育种和研究中国白菜和白菜的基因编辑能力.

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/Cas9是一种强大的基因组编辑工具,但受到原空间细胞相邻基因 (PAM) 识别的限制.
工程 Cas9 变体 (Cas9-NG,SpG,SpRY) 识别非正规的 PAM,扩大了目标范围.
之前的研究集中在米等单一植物上;在布拉西卡蔬菜中的应用仍未得到充分探索.

研究的目的:

为了评估中国白菜 (Brassica rapa) 和白菜 (Brassica oleracea) 中的Cas9-NG,SpG和SpRY变体的基因组编辑效率.
评估这些变种在针对布拉西卡基因组内的非正规PAM位点方面的能力.
开发和测试一种使用SPRY用于Brassica物种的腺基编辑器.

主要方法:

将Cas9-NG,SpG和SpRY系统引入中国的白菜和白菜,通过Agrobacterium介导的转化.
在各种 PAM 位点 (NGN,NGC,NGT,NRN,NYN) 进行向突变发生分析.
开发一种使用SpRY和TadA8e除氨酶进行A-G转换的腺基编辑器.

主要成果:

Cas9-NG和SpG在NGN PAMs中显示出高效的突变发生.
在NGC和NGT PAM中,SpG表现优于Cas9-NG.
通过一些自我定位,SpRY实现了广泛的PAM准 (NRN>NYN),并启用了在白菜中减少PAM的编辑;基于SpRY的腺基编辑器成功诱导了A-to-G转换.

结论:

评估的Cas9变种显著扩大了Brassica蔬菜中的基因组编辑能力.
这些工具有助于针对性的突变和基因改进在经济上重要的巴西卡作物.
这项研究提供了新的基因组编辑工具箱,用于未来的中国白菜和白菜的研究和育种.

关键词:

这就是Brassica Brassica.克里斯普尔是什么意思?克里斯普尔是什么意思?在Cas9的变种中,Cas9的变种.基因组编辑基因组编辑非正规的PAM是没有规范性的.

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