DOI: 10.5061/dryad.nk98sf83j
title: Genomic profile of gene edited hematopoietic stem cells

description: 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&gt;C, which is
 linked to high HbF levels, and -158C&gt;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&gt;C and
 -158C&gt;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&gt;C and -158C&gt;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.  
description: 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  
description: # Genomic profile of gene edited hematopoietic stem cells ## Description
 of the data and file structure 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. ### Files and variables
 #### File: Chandraprabha\_P\_et\_al.zip **Description:** The zip file
 contains folders corresponding to figure numbers as in the article, i.e,
 1A, 2B etc. Each folder contains another folder within it named Sanger
 trace files, in order to describe the content within each sub-folder. And
 every such folder contains the Sanger sequencing trace files of samples of
 respective figures in the article, and each sample ab1 file is labelled
 similar to the label given in the data figure (X-axis). Each folder also
 contains a ‘Control’ ab1 file, which can be used as the unedited reference
 for analysing the results. Detailed description of each folder is as
 follows, described in the order as found within the zip file: **Figure 2F
 -** Subfolders 1_HSPCs: Control and sample (HSPCs) ab1 files for HSPCs
 plot on Figure 2F 2_Colonies: Control and CFU colonies sample (C1-C26) ab1
 files for Colonies plot on Figure 2F **Figure 2I -** Control and sample
 (Labelled similar to figure 2I labels described in coloured dots on left)
 ab1 files for plot Figure 2I; 175, 175 158, B4, 158. Each duplicate per
 sample is numbered 1, 2 etc.  **Figure 3B** – Control (1 & 2) and
 sample (Labelled similar to figure 3B labels described in X-axis) ab1
 files for plot Figure 3B; DMSO, RS-1, RUS, Alt-R, Q-VD-OPH, Cyclosporin-H,
 Eltrombopag, SB-203580, Ursolic acid, Zeatin, Zeatin riboside, XL-413,
 Aphidicolin, Nicotinamide, Hydroxyurea, SCR-7, NU-7441, Trichostatin-A,
 Valproic acid, Entinostat, Panobinostat, Nutlin-3a, Pfithrin-µ,
 Pfithrin-α, UNC-2170. Each duplicate per sample is numbered 1, 2 etc.
  **Figure 3C** – Subfolders - Experiment 1 & Experiment 2
 (Experimental duplicates) Both folders contain Control and sample
 (Labelled similar to figure 3C labels described in X-axis) ab1 files for
 plot Figure 3C; DMSO, NU-7441, AZD-7648, DMNB, LTURM 34, NU-7026. Each
 duplicate per sample is numbered 1, 2 etc.  **Figure 3E** – Subfolders -
 Experiment 1 & Experiment 2 (Experimental duplicates) Both folders
 contain Control and sample (Labelled similar to figure 3E labels described
 in X-axis) ab1 files for plot Figure 3E; 0 µM, 7 µM, and 20 µM for both
 Vehicle and RUS samples. Each duplicate per sample is numbered 1, 2 etc.
 **Figure 4F** – Subfolders – Input (Refers to input genotype of infused
 cells) & In-vivo (Refers to genotype of transplanted cells at 16th
 week)   Both folders contain respective Control and sample (Labelled
 similar to figure 4F labels described in X-axis) ab1 files for plot Figure
 4F; Input; B4, B4-AZD-7648 In-vivo; B4, B4-AZD-7648 for mouse numbers 1-3
 and 2-4, respectively. **Figure 5B& 5C** – Folder contains Control and
 sample (Labelled similar to figure 5B labels described in X-axis) ab1
 files for plot Figure 5B; Control and β-thal model. **Figure 5D** – Folder
 contains respective Control (Labelled as Wild type) and sample (Labelled
 similar to figure 5D labels described in X-axis) ab1 files for plot Figure
 5D; Wild type, β-thal model_Control, β-thal model_158 C to T, β-thal
 model_B4 **Figure 6B** – Subfolders - Experiment 1 & Experiment 2
 (Experimental duplicates) Both folders contain Control and sample
 (Labelled similar to figure 6B labels described in X-axis) ab1 files for
 plot Figure 6B; SC, BM-MSC, WJ-MSC, SC+AZD-7648, BM-MSC+AZD-7648,
 WJ-MSC+AZD-7648 Each duplicated sample is numbered 1, 2,3 etc. **Figure
 7A** – Subfolders (different targets) – -115 HBG promoter -175 & -158
 BCL11A enhancer All three folders contain Control and sample (labelled as
 per sample details in figure 7A and the folder name) ab1 files for plot
 7A; 115HBG, 175158, BCL11Aenh. Respective controls are provided separately
 in each folder. Each duplicated sample is numbered 1, 2,3 etc.
 **Supplementary Figure 1B** – Control and sample (labelled as per sample
 details in figure supplementary 1B) ab1 files for plot supplementary 1B;
 B2, B4. 3 controls are provided corresponding to multiple donor cells used
 in duplicates 1,2,3 Each duplicated sample is numbered 1, 2,3 etc.
 **Supplementary Figure 2C** – Control and sample (labelled as per sample
 details in supplementary figure 2C) ab1 files for plot supplementary 2C;
 RNP, DMSO, RUS. 3 controls are provided corresponding to multiple donor
 cells used in duplicates 1,2,3 Each duplicated sample is numbered 1, 2,3
 etc. **Supplementary Figure 2D** – Subfolders (different targets) –
 Donor_1 Donor_2 Donor_3 All three folders contain Control and sample
 (labelled as per sample details in supplementary figure 2D) ab1 files for
 plot supplementary 2D; CD34+ cells, CD34+CD90+ cells, CD34+CD90- cells
 (for donor 2 CD34+CD90- cells didn’t generate quality reads for analysis,
 so excluded). Respective controls are provided separately in each folder.
 Each duplicated sample is numbered 1, 2,3 etc. **Supplementary Figure 2F**
 – Control and sample (labelled as per sample details in supplementary
 figure 2F) ab1 files for plot supplementary 2F; 3 Controls (T1-T7:
 Genotype at different hours as in figure) T1- 0.5 hours T2- 1 hour T3- 2
 hours T4- 3 hours T5- 6 hours T6- 12 hours T7- 48 hours **Supplementary
 Figure 2G** – Control and sample (labelled as per sample details in
 supplementary figure 2G) ab1 files for plot supplementary 2G; Control
 (T1-T8: Genotype at different hours as in figure) T1- 0.5 hours T2- 1 hour
 T3- 2 hours T4- 3 hours T5- 6 hours T6- 12 hours T7- 24 hours T7- 48 hours
 **Supplementary Figure 3B** – Control and sample (labelled as per sample
 details in figure supplementary 3B) ab1 files for plot supplementary 3B;
 DMSO, 6 hours, 12 hours, 18 hours, 24 hours Each duplicated sample is
 numbered 1, 2 etc. ## Code/software ·         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/#/](https://ice.synthego.com/#/))
 ·         Base editing efficiency was analysed using EditR software
 ([http://baseeditr.com/](http://baseeditr.com/)) ·         Graphs were
 plotted using GraphPad Prism v.10.2 ·         Statistical analyses were
 performed using GraphPad Prism v.10.2