Genome-wide analysis of off-target CRISPR/Cas9 activity in single-cell-derived human hematopoietic stem and progenitor cell clones

Richard H. Smith, Yun Ching Chen, Fayaz Seifuddin, Daniel Hupalo, Camille Alba, Robert Reger, Xin Tian, Daisuke Araki, Clifton L. Dalgard, Richard W. Childs, Mehdi Pirooznia, Andre Larochelle*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

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.

Original languageEnglish
Article number1501
Pages (from-to)1-16
Number of pages16
JournalGenes
Volume11
Issue number12
DOIs
StatePublished - Dec 2020
Externally publishedYes

Keywords

  • CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)
  • Off-target activity
  • Whole genome sequencing (WGS)

Fingerprint

Dive into the research topics of 'Genome-wide analysis of off-target CRISPR/Cas9 activity in single-cell-derived human hematopoietic stem and progenitor cell clones'. Together they form a unique fingerprint.

Cite this