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| Status |
Public on Jun 30, 2023 |
| Title |
Cellular Indexing of Transcriptomes and epitopes sequencing (CITE-seq) analysis to investigate the impact of granulocyte-colony stimulating factor on CRISPR/Cas9 gene edited human hematopoietic stem cell function |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Our research has demonstrated that G-CSF impedes engraftment of CRISPR-Cas9 gene edited human hematopoietic stem cells (HSCs) by exacerbating p53-mediated DNA damage response. Results in this study suggest that the potential for G-CSF to exacerbate HSC toxicity mediated by DNA-damaging nucleases should be considered in autologous HSC gene therapy clinical trials.
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| Overall design |
Human CD34+ cells edited with Cas9 alone (Cas9 group), Cas9+AAVS1-targeting gRNA (RNP group), or Cas9+AAVS1-targeting gRNA+GSE56 (RNP/GSE56 group) were transplanted into busulfan-conditioned NSG mice, and these mice subsequently received 3 days of PBS or G-CSF. Then, each mouse was euthanized and harvested for bone marrow cells and human CD34+cells were isolated using immunomagnetic beads. These CD34+ cells from a total of 6 mice were individually stained with Total seq B CITE-seq antibodies (CD38, CD45RA, CD90, CD49f) + hashtag oligo antibodies 1-6. Then, FACS sort was used to purify ~2,500 cells from each group, and subsequently pooled for CITE-seq library preparation/sequencing. Seurat was used to demultiplex data into 6 individual groups, and differential gene expression analysis was performed to evaluate the impact of G-CSF within Cas9, RNP and RNP/GSE56 groups.
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| Contributor(s) |
Larochelle A, Redekar N, Chen V |
| Citation missing |
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| Submission date |
Apr 20, 2023 |
| Last update date |
Jun 30, 2023 |
| Contact name |
Daisuke Araki |
| E-mail(s) |
daisuke.araki@nih.gov
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| Phone |
301-827-1467
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| Organization name |
National Institutes of Health
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA957792 |
