对Cas9编辑和转录组的联合单细胞分析揭示了广泛的非目标事件和对基因表达的影响
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Updated: Sep 9, 2025
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
855
bioRxiv : the preprint server for biology
|September 5, 2025
概括
在单细胞分辨率下精确测量CRISPR-Cas9基因编辑结果和效果. 这一进步揭示了影响基因表达的广泛的Cas9非目标编辑, 对于基因组工程应用至关重要.
科学领域:
- 基因组学和分子生物学
- 基因编辑技术
- 单细胞分析
背景情况:
- 在单细胞分辨率下测量编辑结果和功能影响方面,CRISPR-Cas9基因组工程面临挑战.
- 准确评估目标和目标之外的编辑对于推进CRISPR-Cas9应用至关重要.
研究的目的:
- 推出Superb-seq,这是一种用于在单细胞分辨率下联合测量Cas9的上位和下位编辑及其基因表达效应的新技术.
- 在一个大规模的细胞实验中描述Cas9非目标编辑的流行程度和影响.
主要方法:
- 超级seq使用T7在位转录和单细胞RNA测序.
- 用于10,000个K562细胞,针对4个染色体重塑基因和7个导向RNA.
- 联合测量Cas9编辑和下游基因表达变化.
主要成果:
- 在目标和36个目标以外的6,230个细胞中,Superb-seq确定了11,891个编辑事件.
- 七个高精度指导中的六个引发了可检测的非目标编辑.
- 在USP9X中发生的一次非目标编辑导致了广泛的下游基因表达中断.
结论:
- Cas9的脱是普遍的,经常发生在内部,并可能对基因表达产生广泛的影响.
- 超级seq是一种多功能,可访问的技术,可实现全基因组CRISPR屏幕和基因编辑指南的功能特征.
- 这项技术克服了评估CRISPR-Cas9编辑结果和功能后果的先前限制.
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