Genome editing for human gene therapy.

Meissner TB1, Mandal PK2, Ferreira LM1, Rossi DJ3, Cowan CA4.
Source: Methods Enzymol
Publication Date: (2014)
Issue: 546: 273-95
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b

Nucleofection of primary human CD4 positive T cells and human mobilized CD34 cells with the Lonza ready-to-use protocols and the suggested programs. Excellen FACS data for purity of the cells and transfection results. Co-transfection of CRISPR/Cas9 complexes. 1-5 million cells, 5 µg Cas9-2A-GFP plasmid, 2.5 µg Guide plasmid#1
2.5 µg Guide plasmid#2, 100 µl Human CD4+ T cell. 


The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. In this chapter, we explore the use of the CRISPR/Cas9 system for the efficient ablation of genes in two clinically relevant primary human cell types, CD4+ T cells and CD34+ hematopoietic stem and progenitor cells. By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.