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

Summary, in English

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: Pankaj K Mandal; Leonardo M R Ferreira; Ryan Collins; Torsten B Meissner; Christian L Boutwell; Max Friesen; Vladimir Vrbanac; Brian S Garrison; Alexei Stortchevoi; David Bryder; Kiran Musunuru; Harrison Brand; Andrew M Tager; Todd M Allen; Michael E Talkowski; Derrick J Rossi; Chad A Cowan
• Department/s: Department of Experimental Medical Science; StemTherapy: National Initiative on Stem Cells for Regenerative Therapy
• Publishing year: 2014
• Language: English
• Pages: 643-652
• Publication/Series: Cell Stem Cell
• Volume: 15
• Issue: 5
• Links: http://www.ncbi.nlm.nih.gov/pubmed/25517468?dopt=Abstract; http://dx.doi.org/10.1016/j.stem.2014.10.004
• Document type: Journal article
• Publisher: Cell Press
• Topic: Cell Biology
• Status: Published
• ISBN/ISSN/Other: ISSN: 1934-5909