对于基因淘汰的CRISPR/Cas9载体构造
Markus Freudhofmaier1, Jacob W Hoyle2,3, Fatemeh Maghuly4
1Plant Functional Genomics Lab, Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria. markus.freudhofmaier@boku.ac.at.
Methods in molecular biology (Clifton, N.J.)
|April 24, 2024
概括
本协议详细介绍了一种具有成本效益的方法,用于制造植物CRISPR/Cas9等离子体. 它通过使用简单的克隆技术组装指导RNA表达结构来实现高效的基因组编辑.
科学领域:
- 植物生物技术 植物生物技术
- 分子生物学分子生物学
- 基因组编辑 基因组编辑
背景情况:
- 克里斯普尔/卡斯9技术允许有针对性的DNA修饰.
- 有效的植物转化需要专门的等离子体.
- 非同源端结合 (NHEJ) 是一个关键的DNA修复途径.
研究的目的:
- 描述一种节约资源的协议,用于构建植物转化等离子体.
- 为了促进CRISPR/Cas9介导的植物基因组编辑.
主要方法:
- 使用Medicago truncatula U6.6促进剂和支架序列组装导向RNA (gRNA) 表达盒.
- 针对特定目标的寡核酸片段的合成.
- 无结合克隆和PCR放大用于gRNA结构生成.
- 将gRNA结构集成到表达Cas9核酶的植物转化载体中.
主要成果:
- 一种简化方法,用于制造表达Cas9和gRNA的植物转化等离子体.
- 使用易于获得的试剂和寡核酸的经济有效的方法.
- 促进诱导双链断裂 (DSB) 用于基因组编辑.
结论:
- 该协议为在植物中进行CRISPR/Cas9基因组编辑的研究人员提供了明确有效的策略.
- 该方法降低了成本,并简化了基本转换向量的创建.
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