Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

ABSTRACT: CRISPR-Cas base editors are preferred tool for genome editing as they generate desired editing without any double strand break in the genome, as double stand break is detrimental to the cells. In our study we have demonstrated the significance of base editors in editing the highly homologous HBG promoter (HBG1 and HBG2) region to introduce novel HPFH-like mutation to elevate HbF for therapeutical applications. Previous studies revealed that the base editors can cause unintended Cas-independent edits at transcriptome level. To validate off-target at RNA level, we performed a transcriptome wide analysis. The frequency of unintended edits in the HUDEP-2 stable cell lines expressing the base editors with the gRNA were not significant compared to the control. We determined the ABE mediated A to I conversion and CBE mediated C to U conversion across the base edited samples. The RNA off-target analysis was carried out with the help of REDItools v 2 tool. This data suggests that despite high on-target editing in DNA, the Cas-independent RNA off-target were not at detectable range compared to control. The differential expression of 34 selected genes which necessitate globin regulation were compared between the unedited HUDEP WT, CBE control, ABE control, and edited ABE (with gRNA 2/11) and edited CBE (with gRNA 2/11). We observed that there is no significant differential gene expression between the edited and control cells except the gamma and delta globin genes. These results suggest that base editors are preferred tools to edit highly homologous HBG promoter region to created HPFH-like mutations inducing HbF levels without causing double strand breaks, larger deletions and no significant RNA off-targets which are detrimental to the gene edited cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE192801 | GEO | 2022/01/02

REPOSITORIES: GEO