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Gene therapy is a technique where a gene is inserted into a person’s cells to prevent or treat a serious disease. The added gene may be a healthy version of the gene that is mutated in the patient, or it could be a different gene that inactivates or compensates for the patient’s disease-causing gene. For example, in patients with severe combined immunodeficiency (SCID) due to a mutation in the gene for the enzyme adenosine deaminase, a functioning version of the gene can be...

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Related Experiment Video

Updated: Apr 20, 2026

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Published on: February 2, 2016

Genome editing for human gene therapy.

Torsten B Meissner¹, Pankaj K Mandal², Leonardo M R Ferreira¹

¹Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

Methods in Enzymology

|November 16, 2014

Summary

This summary is machine-generated.

CRISPR/Cas9 gene editing efficiently deletes target genes in human hematopoietic cells. This dual guide RNA strategy enables precise gene ablation for potential gene therapy applications in genetic disorders.

Keywords:

CD34+ HSPCs CD4+ T Cells CRISPR/Cas9 Dual guide strategy Gene therapy Genome editing

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Published on: March 5, 2017

Area of Science:

Molecular Biology
Gene Therapy
Hematology

Background:

Genome-editing technologies like CRISPR/Cas9 offer precise genetic manipulation.
Human gene therapy aims to treat genetic disorders using advanced tools.
Hematopoietic stem cells are crucial for treating blood-related genetic diseases.

Purpose of the Study:

To investigate CRISPR/Cas9 for efficient gene ablation in primary human hematopoietic cells.
To assess the feasibility of dual guide RNA strategy for gene deletion.
To explore applications in treating hemoglobinopathies, immunodeficiencies, and cancer.

Main Methods:

Utilized the CRISPR/Cas9 system with dual guide RNAs for targeted gene deletion.
Employed a Cas9-2A-GFP fusion protein for FACS-based cell enrichment.
Tested efficiency in CD4+ T cells and CD34+ hematopoietic stem and progenitor cells.

Main Results:

Achieved highly efficient and predictable gene deletions using the dual guide RNA approach.
Demonstrated successful gene ablation in clinically relevant primary human cell types.
Facilitated FACS-based enrichment of successfully transfected cells.

Conclusions:

The dual guide RNA CRISPR/Cas9 strategy is effective for gene ablation in human hematopoietic cells.
This method shows promise for developing gene therapies for genetic blood disorders.
CRISPR/Cas9 offers a versatile tool for gene therapy development.