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Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

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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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

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.

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HiPSC
Neural Lineage
Gene Targeting
CRISPR Cas9
Cas9 Double Nickase
Homologous Recombination
Reporter Lines
Genetic Manipulation
Genome Editing

Chapters in this video

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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