Genomic profile of gene edited hematopoietic stem cells

Sickle cell disease (SCD) and β-thalassemia patients with elevated gamma globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this natural phenomenon, the most coveted gene therapy approach is to edit the regulatory sequences of HBG1/G2 to reactivate them, with a magnified effect by simultaneous targeting of multiple sequences. Here, we used Cas9 RNP-ssODN-mediated homology-directed gene editing to mimic two naturally occurring HBG promoter point mutations, namely, -175T>C, which is linked to high HbF levels, and -158C>T, the most common polymorphism in the Indian population that induces HbF under erythropoietic stress, in HSPCs. We observed high complete HDR conversions, with at least 30% of HSPCs exhibiting both -175T>C and -158C>T mutations, which increased to over 50% under optimized conditions. In NBSGW mice, up to 30% of long-term engrafted human HSPCs showed both -175T>C and -158C>T HDR conversions, with the efficiency peaking with up to 57% of HSPCs containing at least one of the beneficial mutations. Cas9 RNP-ssODN-based nucleotide conversion at the HBG promoter offers a promising gene therapy approach to ameliorate the disease phenotypes of both β-thalassemia and SCD. The developed approach can simplify and broaden applications that require multiple nucleotide modifications in HSPCs.

Methods
The dataset includes chromatogram trace files obtained from Sanger sequencing. The Genotyping analysis was carried out as described below:

Genomic DNA extraction was performed 3-5 days post nucleofection utilizing QuickExtract™ DNA Extraction Solution. Over 10,000 cells were collected and rinsed with 1X PBS. The cell pellet was then resuspended in approximately 20-50 µl of QuickExtract™ solution and subjected to sequential incubation at 68°C and 98°C. The extracted DNA was directly processed for amplification of the target using the appropriate primers. Following the confirmation of bands through gel electrophoresis, the amplified product was purified using the Macherey-Nagel™ NucleoSpin™ PCR Clean-up Kit. For Sanger sequencing, the purified PCR amplicon was processed with BigDye™ Terminator v3.1 Cycle Sequencing. The resulting chromatogram traces are provided in this dataset.

Sequences ab1 files are  viewed using ab1 trace files can be viewed using Snapgene sequence viewer. Editing efficiency for samples was analysed using Synthego ICE analysis : Inference of CRISPR edits (https://ice.synthego.com/#/). Base editing efficiency was analysed using EditR software (http://baseeditr.com/). Graphs were plotted using GraphPad Prism v.10.2 .Statistical analyses were performed using GraphPad Prism v.10.2