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

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.

A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

“…With the widespread interest in gene editing as a new therapeutic modality, concerns about hazardous on- and off-target mutations in edited cell products have become more visible (Sheridan, 2021). A string of recent reports have uncovered previously underappreciated lesions, such as kilo- and megabase-scale deletions as well as chromotrypsis, illustrating the potential risk of Cas9-based gene editing (Adikusuma et al, 2018; Boutin et al, 2021; Leibowitz et al, 2021). Our defined culture system addresses this concern in the murine model by enabling marker-free selection of edited LT-HSC clones with known on- and off-target mutational profiles.…”

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

“…Until now, investigation of the potential source of genotoxicity during gene editing mainly focused on off-target nuclease activity and the occurrence of genomic rearrangements at the target site ( Adikusuma et al., 2018 ; Boutin et al., 2021 ; Kosicki et al., 2018 ; Lattanzi et al., 2021 ; Leibowitz et al., 2021 ; Nahmad et al., 2022 ; Turchiano et al., 2021 ). The genotoxic risk ascribed to DNA template delivery, its persistence, and its integration in edited cells ( Colella et al., 2018 ; Penaud-Budloo et al., 2018 ; Schnödt and Büning, 2017 ) remains poorly investigated, especially for HSPCs.…”

Modern therapeutic approaches to liver-related disorders

“…30 Finally, the major caveats are potential genotoxicity, possible off-target effects, and large-scale chromosomal deletions. 89,90 Safety data are scarce in human patients, and toxicity will need to be monitored closely.…”