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CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

JoVE Revista
Genética

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JoVE Revista Genética

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Please note that all translations are automatically generated. Click here for the English version.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

DOI:

10.3791/56195-v

•

10:07 min

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August 25, 2017

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Natalia Rivera-Torres², Eric B. Kmiec²

¹Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute,Christiana Care Health Services, ²Department of Medical Sciences,University of Delaware

Capítulos

00:05Título
00:38Cell Line, Culture Conditions, and Harvest
01:57Plating the Cells and Releasing Them from Aphidicolin Synchronization
02:44RNA Complexing
04:13Harvesting the Cells for Targeting
05:30Targeting Samples
06:13Analysis of Gene Edited Cells and Transfection Efficiency
06:57DNA Sequence Analysis
07:56Results: Effective Point Mutation Repair
09:41Conclusion

Summary

Traducción Automática

该协议概述了 CRISPR/Cas9-based 基因编辑系统的工作流程, 用于修复哺乳动物细胞的点突变。在这里, 我们使用一个组合的方法来基因编辑和详细的后续实验策略, 测量 indel 形成在目标 site-in 本质, 分析现场诱变。

Tags

CRISPR/Cas9 SsODN On-site Mutagenesis Homology Directed Repair Gene Correction HTT116 Cells Trypan Blue Hemocytometer Aphidicolin EGFP Gene Guide Sequences CrRNA TracrRNA

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