Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.

Cell Stem Cell
Authors
Keywords
Abstract

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.

Year of Publication
2014
Journal
Cell Stem Cell
Volume
15
Issue
5
Pages
643-52
Date Published
2014 Nov 06
ISSN
1875-9777
URL
DOI
10.1016/j.stem.2014.10.004
PubMed ID
25517468
PubMed Central ID
PMC4269831
Links
Grant list
R01HL107630 / HL / NHLBI NIH HHS / United States
U01 HL100408 / HL / NHLBI NIH HHS / United States
P01-AI104715 / AI / NIAID NIH HHS / United States
R00 MH095867 / MH / NIMH NIH HHS / United States
K99 MH095867 / MH / NIMH NIH HHS / United States
U01DK072473 / DK / NIDDK NIH HHS / United States
U01 DK072473 / DK / NIDDK NIH HHS / United States
R01 HL107630 / HL / NHLBI NIH HHS / United States
U01HL100408 / HL / NHLBI NIH HHS / United States
P01 AI104715 / AI / NIAID NIH HHS / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
U01 HL107440 / HL / NHLBI NIH HHS / United States
R01DK097768 / DK / NIDDK NIH HHS / United States
MH095867 / MH / NIMH NIH HHS / United States
P30 AI060354 / AI / NIAID NIH HHS / United States