相关实验视频
Updated: Jul 23, 2025
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
690
通过MMEJ介导的CRISPR基因组编辑的长读序列分析揭示了复杂的目标载体插入,这些插入可能会逃避基于PCR的标准质量控制
Yuki Higashitani1, Kyoji Horie2
1Department of Physiology II, Nara Medical University, Kashihara, Nara, 634-8521, Japan.
Scientific reports
|July 19, 2023
概括
通过CRISPR基因组编辑,可以通过多个重组途径导致意外的载体插入. 严格的质量控制对于识别复杂的,准确的插入至关重要,并确保精确的基因编辑结果.
科学领域:
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
- 生物技术是生物技术.
背景情况:
- 克里斯普尔基因组编辑对于理解基因功能至关重要.
- 精确的基因组修改依赖于理解重组机制.
- 微同质介导端结合 (MMEJ) 是一个关键的CRISPR相关修复途径.
研究的目的:
- 为了研究在CRISPR-Cas9中介生成条件等位基因的过程中复杂的载体插入.
- 确定导致意外插入的重组机制.
- 突出基因组编辑中强有力的质量控制的必要性.
主要方法:
- 使用了CRISPR-Cas9与含有loxP位点和微同质的准向量.
- 采用PCR查来检测小鼠胚胎干细胞中的重组事件.
- 应用纳米孔测序来表征意想不到的DNA带.
- 分析了异构的单核酸多态性,以评估等位基因修饰.
主要成果:
- 观察到频繁的,大于预期的PCR波段,表明复杂的插入.
- 纳米孔测序揭示了由MMEJ介导的插入,非同源端连接和同源重组.
- 意想不到的等位基因逃脱了标准PCR查,需要改进检测方法.
- 在分析的克隆中,至少有17%的克隆表现出复杂的,针对目标的矢量插入.
结论:
- 通过各种重组途径,CRISPR编辑可以导致复杂的,无意的对目标矢量插入.
- 标准选方法可能无法检测所有意想不到的修改.
- 彻底的质量控制,包括先进的测序和基因组特异性分析,对于验证基因组编辑克隆至关重要.
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