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

Mandal, Pankaj K; Ferreira, Leonardo M R; Collins, Ryan; Meissner, Torsten B; Boutwell, Christian L; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S; Stortchevoi, Alexei and Bryder, David LU , et al. (2014) In Cell Stem Cell 15(5). p.643-652
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... (More)
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. (Less)
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Cell Stem Cell
volume
15
issue
5
pages
643 - 652
publisher
Cell Press
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  • pmid:25517468
  • wos:000345012700016
  • scopus:84922671463
ISSN
1934-5909
DOI
10.1016/j.stem.2014.10.004
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English
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yes
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cb0c1060-2f23-4ba2-bc1f-b88cba5208a9 (old id 4908074)
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http://www.ncbi.nlm.nih.gov/pubmed/25517468?dopt=Abstract
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2015-01-07 15:51:44
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@article{cb0c1060-2f23-4ba2-bc1f-b88cba5208a9,
  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.},
  author       = {Mandal, Pankaj K and Ferreira, Leonardo M R and Collins, Ryan and Meissner, Torsten B and Boutwell, Christian L and Friesen, Max and Vrbanac, Vladimir and Garrison, Brian S and Stortchevoi, Alexei and Bryder, David and Musunuru, Kiran and Brand, Harrison and Tager, Andrew M and Allen, Todd M and Talkowski, Michael E and Rossi, Derrick J and Cowan, Chad A},
  issn         = {1934-5909},
  language     = {eng},
  number       = {5},
  pages        = {643--652},
  publisher    = {Cell Press},
  series       = {Cell Stem Cell},
  title        = {Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9.},
  url          = {http://dx.doi.org/10.1016/j.stem.2014.10.004},
  volume       = {15},
  year         = {2014},
}