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

Shenglan Li; Haipeng Xue; Bo Long; Li Sun; Tai Truong; Ying Liu

doi:10.3791/52539

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

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

10.5K views

⸱

DOI:

10.3791/52539

⸱

May 28th, 2015

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

^* These authors contributed equally

Summary

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.

Abstract

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Introduction

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, together with other genome editing tools, has revolutionized the human genome manipulation in recent years^1-7. Major components in the CRISPR/Cas9 system are single guide RNA (sgRNA) and Cas9. Cas9 is an RNA-guided type II DNA endonuclease. It has two nuclease domains, HNH and RuvC, which are responsible for making DNA double-stranded breaks (DSBs) at a specific genomic region next to a protospacer adjacent motif (PAM)^7-10.An sgRNA has a combined function of that of crRNA and tracrRNA, two adaptive immunity RNA molecules identified in bacteria (such as Streptococcus....