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 H..., 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 eff..., , # 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.

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# 基因编辑造血干细胞的基因组图谱

## 数据及文件结构描述

镰状细胞病（Sickle cell disease, SCD）和β-地中海贫血患者若γ珠蛋白（HBG1/G2）水平升高，则表现出轻微症状或无症状。为重现这一自然现象，最理想的基因治疗策略是编辑HBG1/G2的调控序列以重新激活它们，同时靶向多个序列可增强效果。本研究中，我们利用Cas9 RNP-ssODN介导的同源定向基因编辑（homology-directed gene editing）在造血干细胞和祖细胞（hematopoietic stem and progenitor cells, HSPCs）中模拟两种自然发生的HBG启动子点突变：其一为与高胎儿血红蛋白（fetal hemoglobin, HbF）水平相关的-175T>C突变，其二为印度人群中最常见的多态性-158C>T突变，该突变可在造血应激条件下诱导HbF表达。我们观察到较高的完全同源定向修复（homology-directed repair, HDR）转化率，至少30%的HSPCs同时携带-175T>C和-158C>T突变，优化条件下该比例可提升至50%以上。在NBSGW小鼠中，长期移植的人类HSPCs中高达30%同时存在-175T>C和-158C>T的HDR转化，效率最高可达57%的H...。本数据集包含通过桑格测序（Sanger sequencing）获得的色谱图轨迹文件。基因分型（Genotyping）分析方法如下：

基因组DNA提取在核转染后3-5天进行，使用QuickExtract™ DNA提取溶液。收集超过10000个细胞并用1X PBS冲洗。随后将细胞沉淀重悬于约20-50 µl QuickExtract™溶液中，并依次在68°C和98°C下孵育。提取的DNA直接用适当引物进行靶标扩增。通过凝胶电泳确认条带后，使用Macherey-Nagel™ NucleoSpin™ PCR纯化试剂盒纯化扩增产物。桑格测序时，纯化的PCR扩增子用BigDye™ Terminator v3.1循环测序试剂盒处理。本数据集提供了所得的色谱图轨迹文件。

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