CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

Nat Commun. 2021 Aug 13;12(1):4922. doi: 10.1038/s41467-021-25190-6.

Abstract

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Chromosome Deletion
  • Chromosomes, Human, Pair 11 / genetics
  • DNA Methylation
  • Gene Editing / methods*
  • Gene Expression
  • Globins / genetics*
  • HEK293 Cells
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Insulin-Like Growth Factor II / genetics
  • Loss of Heterozygosity / genetics*
  • Polymorphism, Single Nucleotide
  • RNA, Long Noncoding / genetics
  • Sequence Deletion*

Substances

  • H19 long non-coding RNA
  • RNA, Long Noncoding
  • Insulin-Like Growth Factor II
  • Globins