Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones

Genes (Basel). 2020 Dec 13;11(12):1501. doi: 10.3390/genes11121501.


CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.

Keywords: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9); off-target activity; whole genome sequencing (WGS).

Publication types

  • Clinical Trial
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • CRISPR-Cas Systems*
  • Chromosomes, Human, Pair 19 / genetics*
  • Chromosomes, Human, Pair 2 / genetics*
  • Clone Cells*
  • Female
  • Gene Editing*
  • Genetic Loci
  • Hematopoietic Stem Cells*
  • Humans
  • Receptors, CXCR4 / genetics


  • CXCR4 protein, human
  • Receptors, CXCR4