Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

Abstract

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34 cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34 cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.

High level of fetal-globin reactivation by designed transcriptional activator-like effector

“…Altogether, these results have proven the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations. 12 CRISPR/Cas9 has a significant rate of off-target mutations, and therefore, it might cause some adverse effects. 13 ZFNs are very difficult to engineer and, in addition, produce greater cell toxicity by inhibiting cell growth.…”

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells

“…Deletion of genomic elements using pairs of Cas9 RNPs is useful for defining regions of phenotypic interest. However, two DSBs in one targeted cell could be resolved to a variety of genotypes besides simple deletion: inversions, duplications, and short insertions or deletions at either cut site [19,32]. To avoid these complications, a single cut site is preferred.…”

Engineering Globin Gene Expression

“…In addition, targeted nucleases have been shown to create unwanted chromosomal rearrangements. This may be due to the presence of sequences of partial homology, as was shown for rearrangements between the δ- and β-globin genes after induction of DSBs at the β-globin gene 70 …”