在体内使用DISCOVER-Seq无偏见地检测CRISPR外标
Beeke Wienert1,2,3, Stacia K Wyman1, Christopher D Richardson1,2
1Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94704, USA.
概括
我们开发了DISCOVER-Seq, 这是一种直接在体内发现CRISPR-Cas基因组编辑异常效应的新方法. 这种方法追踪DNA修复因子MRE11,以精确,单基分解细胞和生物中的Cas活性.
科学领域:
- 分子生物学
- 基因组学
- 生物技术
背景情况:
- CRISPR-Cas基因组编辑精确地准DNA, 但可能导致意外的异位突变.
- 目前用于检测非目标效应的方法仅限于纯化DNA或特定细胞模型,缺乏体内可用性.
- 在体内直接识别非目标部位对于基因组编辑疗法的安全性和有效性至关重要.
研究的目的:
- 开发一种普遍适用的,无偏见的方法,直接在体内识别CRISPR-Cas非目标部位.
- 通过利用DNA修复因子来检测单基分辨率的Cas活性.
- 为了使新编辑工具的特征化,并促进患者特定的目标分析.
主要方法:
- 开发了DISCOVER-Seq (发现现场Cas目标外物和通过测序进行验证),这是一种不偏见的目标外物识别的新方法.
- 使用DNA修复因子MRE11的招募来确定CAS活动和非目标部位.
- 在各种细胞系,患者诱导的多能干细胞和体内小鼠模型中验证了该方法.
主要成果:
- DISCOVER-Seq可以直接在体内检测CRISPR-Cas的目标部位.
- 追踪MRE11的招募提供了单个基础的Cas活动和非目标位置的分辨率.
- 该方法与多种指导RNA格式和Cas酶类型兼容,方便对新的基因组编辑工具进行评估.
- 在细胞系,iPSC和在体内小鼠编辑过程中成功识别了非目标部位.
结论:
- 在基因组编辑中,DISCOVER-Seq为无偏见的体内非目标发现提供了普遍适用的解决方案.
- 这项技术可以精确地描述Cas活性,并识别分子水平上的非目标突变.
- DISCOVER-Seq为个人患者的现场非目标分析铺平了道路,这对于推进治疗基因组编辑安全性和应用至关重要.
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