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

Effiziente Erzeugung von hiPSC Neural Lineage Spezifische Knockin Reporter Mit den CRISPR / Cas9 ...

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JoVE Journal Developmental Biology

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

Effiziente Erzeugung von hiPSC Neural Lineage Spezifische Knockin Reporter Mit den CRISPR / Cas9 und Cas9 Doppel Nickase-System

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14:46 min

May 28, 2015

DOI: 10.3791/52539-v

Shenglan Li*^1,2, Haipeng Xue*^1,2, Bo Long^1,2,5, Li Sun^1,2,6, Tai Truong^1,2,4,7, Ying Liu^1,2,3

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

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Overview

This study presents a protocol for generating lineage-specific human induced pluripotent stem cell (hiPSC) knockin reporters using the CRISPR/Cas9 system. The method enhances gene targeting efficiency through homologous recombination.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Gene Editing

Background

CRISPR/Cas9 is a revolutionary tool for genome editing.
hiPSCs are valuable for studying human development and disease.
Lineage-specific reporters are essential for tracking cell fate.
Homologous recombination improves the precision of gene targeting.

Purpose of Study

To develop a reliable method for creating lineage-specific hiPSC reporters.
To utilize CRISPR/Cas9 for efficient gene editing in hiPSCs.
To facilitate the study of neural lineage differentiation.

Methods Used

Design and construct targeting vectors for gene editing.
Create single guide RNAs for the CRISPR/Cas9 system.
Cotransfect hiPSCs with guide RNAs and Cas9 expression vectors.
Evaluate gene editing efficiency using T7 endonuclease I assay.

Main Results

Successful generation of lineage-specific hiPSC knockin reporters.
Demonstrated high efficiency of CRISPR/Cas9-mediated homologous recombination.
Identified potential off-target sites through in silico analysis.
Established neural lineage reporter cell lines for future studies.

Conclusions

The CRISPR/Cas9 system is effective for generating hiPSC reporters.
This method can advance research in neural development and disease.
Future applications may include studying gene function in neural lineages.

Frequently Asked Questions

What is the CRISPR/Cas9 system?

CRISPR/Cas9 is a genome editing technology that allows for precise modifications of DNA in living organisms.

Why are lineage-specific reporters important?

They enable researchers to track the development and differentiation of specific cell types.

How does homologous recombination improve gene targeting?

It allows for precise insertion of genetic material at specific locations in the genome.

What are the potential applications of this research?

Applications include studying neural development, disease modeling, and regenerative medicine.

What are off-target effects in CRISPR editing?

Off-target effects occur when CRISPR modifies unintended sites in the genome, which can lead to unwanted consequences.

How can off-target effects be minimized?

Careful design of guide RNAs and thorough validation of editing events can help minimize off-target effects.

Genom-Editing-Tools wie das CRISPR-System (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) haben die Effizienz des Gen-Targetings in humanen induzierten pluripotenten Stammzellen (hiPSCs) erheblich verbessert. Dieses Manuskript beschreibt ein Protokoll zur Generierung eines linienspezifischen hiPSC-Reporters unter Verwendung der CRISPR/Cas-System-unterstützten homologen Rekombination.

Das übergeordnete Ziel dieses Verfahrens ist die Generierung von linienspezifischen humanen induzierten pluripotenten Stammzellen oder H-I-P-S-C-Knockin-Reportern unter Verwendung der CRISPR Cas 9-vermittelten homologen Rekombination. Dies wird erreicht, indem zunächst Targeting-Vektoren entworfen und konstruiert werden. Der zweite Schritt besteht darin, Single-Guide-RNAs für das CRISPR Cas nine System oder das CAS nine N Double Nick Case System zu entwerfen und zu konstruieren.

2 93 FUSS. Die Zellen werden dann mit den Single-Guide-RNAs und dem CAS-Neun-Expressionsvektor transfiziert, und die DNA wird extrahiert und für die Auswertung durch einen T-7-ENDONUKLEASE-One-Assay amplifiziert. Der letzte Schritt besteht darin, eine In-silico-Vorhersage potenzieller Off-Target-Stellen durchzuführen. Lagen. Letztendlich werden HI-PSCs mit dem Zielvektor und den neun CRISPR-Cas-Systemkomponenten transfiziert, um Knockin-Reporterzelllinien der neuronalen Abstammung herzustellen.

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Schlüsselwörter: HiPSC neuronale Abstammung Gene Targeting CRISPR/Cas9 Cas9-Doppel-Nickase homologe Rekombination Reporterlinien genetische Manipulation Genome Editing