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

Cell Stem Cell. 2014 Nov 6;15(5):643-52. doi: 10.1016/j.stem.2014.10.004. Epub 2014 Nov 6.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD34 / metabolism
  • CRISPR-Associated Proteins / metabolism*
  • Cell Lineage
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gene Deletion*
  • Gene Targeting
  • Genetic Loci
  • Genome, Human / genetics
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mice
  • RNA Editing / genetics
  • RNA, Guide / metabolism
  • Receptors, CCR5 / metabolism

Substances

  • Antigens, CD34
  • CCR5 protein, human
  • CRISPR-Associated Proteins
  • RNA, Guide
  • Receptors, CCR5

Associated data

  • BioProject/PRJNA264619