Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9

Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL, Friesen M, Vrbanac V, Garrison BS, Stortchevoi A, Bryder D, Musunuru K, Brand H, Tager AM, Allen TM, Talkowski ME, Rossi DJ, Cowan CA
Source: Cell Stem Cell
Publication Date: (2014)
Issue: 15(5): 643-652
Research Area:
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
Species: human
Tissue Origin: blood
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: kidney
Nucleofector® I/II/2b
Human primary CD4+ T cells and CD34+ HSPCs were transfected with Cas9-2A-GFP and gRNA encoding plasmids using respective Amaxa kits (Human CD34 cell Nucleofector kit #VPA-1003 for CD34+ HSPCs, Human T cell Nucleofector kit #VPA-1002 for CD4+ T cells, and Cell Line Nucleofector kit V #VCA-1003 for K562 cells) and cell-specific program (U-008 for CD34+ HSPCs, U-014 for CD4+ T cells, and T-016 for K562 cells) with an Amaxa II device as per manufacturers instructions with minor modifications. For dual gRNA combinations in CD34+ HSPCs, individual gRNAs were used at half the amount of single gRNA conditions, keeping total gRNA amount the same across the experimental settings. Lonza summary: In this study, Mandal et al. efficiently ablated two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells HSPCs through the delivery of CRISPR/Cas9 dual RNA guides with the Nucleofector technology. The modified CD34+ HSPCs retain multi-lineage potential in vitro and in vivo, very high on-target and minimal off target mutation levels. This study paves the way for gene and cell-based therapies of blood diseases.
Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.