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

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.

Ferreira, Leonardo M R (author)
Collins, Ryan (author)
Meissner, Torsten B (author)
Boutwell, Christian L (author)
Friesen, Max (author)
Vrbanac, Vladimir (author)
Garrison, Brian S (author)
Stortchevoi, Alexei (author)
Bryder, DavidLund University,Lunds universitet,Institutionen för experimentell medicinsk vetenskap,Medicinska fakulteten,Department of Experimental Medical Science,Faculty of Medicine(Swepub:lu)med-dbr (author)
Musunuru, Kiran (author)
Brand, Harrison (author)
Tager, Andrew M (author)
Allen, Todd M (author)
Talkowski, Michael E (author)
Rossi, Derrick J (author)
Cowan, Chad A (author)
Institutionen för experimentell medicinsk vetenskapMedicinska fakulteten (creator_code:org_t)

By the author/editor: Mandal, Pankaj K; Ferreira, Leonar ...; Collins, Ryan; Meissner, Torste ...; Boutwell, Christ ...; Friesen, Max; show more...; Vrbanac, Vladimi ...; Garrison, Brian ...; Stortchevoi, Ale ...; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M; Allen, Todd M; Talkowski, Micha ...; Rossi, Derrick J; Cowan, Chad A; show less...

About the subject

NATURAL SCIENCES: NATURAL SCIENCES; and Biological Scien ...; and Cell Biology

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