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的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

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11.0K Views

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14:46 min

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May 28th, 2015

DOI :

10.3791/52539-v

May 28th, 2015

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Shenglan Li*¹^,², Haipeng Xue*¹^,², Bo Long¹^,²^,⁵, Li Sun¹^,²^,⁶, Tai Truong¹^,²^,⁴^,⁷, Ying Liu¹^,²^,³

¹Department of Neurosurgery, The University of Texas Health Science Center at Houston, ²Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, ³The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, ⁴Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, ⁵Department of Anesthesiology, Shengjing Hospital, China Medical University, ⁶Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, ⁷Biology Department, University of West Georgia

摘要

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

探索更多视频

Cas9 Double Nickase

Homologous Recombination

Genetic Manipulation

此视频中的章节

0:05

Title

1:54

Design of Targeting Vectors

2:41

Design and Vector Construction of sgRNAs for CRISPR/Cas9 System

5:20

Design and Construction of Cas9n Double Nickase sgRNA

8:06

Evaluation of sgRNAs by T7 Endonuclease1

11:03

In Silico Prediction of Potential Off Target Sites

12:32

Results: The T7E1 Assay is Used to Evaluate sgRNAs Prior to Transfecting hiPSCs

13:56

Conclusion

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