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通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率
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Bioengineering
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JoVE Journal Bioengineering
Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System
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通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率

DOI:
10.3791/59639-v

•

05:51 min

•

December 05, 2020

•

, , , , , ,
1Qingdao Agricultural University, Qingdao, China, 2University of Cantabria, Santander y Torrelavega, Spain

Chapters

  • 00:05Introduction
  • 00:45Oligonucleotide Annealing and sgRNA/CRISPR Vector Digestion
  • 01:51Identification of the Correct Recombinant Plasmids by PCR
  • 02:42Dual-luciferase Detection
  • 04:16Results: Design of sgRNA to Target Sheep DKK2 Exon 1
  • 05:20Conclusion

Summary

Automatic Translation

Automatic Translation

在这里，我们提出了一个协议，描述了一个简化的方法，用于有效生成质粒，同时表达CRISPR酶和相关的单导RNA（sgRNA）。哺乳动物细胞与这种sgRNA/CRISPR载体的共转染，以及检查双链断裂修复的双荧光素酶报告器载体，可以评估敲除效率。

Tags

CRISPRPlasmid ConstructionDual Luciferase Reporter SystemSgRNAGene EditingPCRCell TransformationBacterial ColonyRecombinant PlasmidsCell LysisDual Luciferase Assay

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