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

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. 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.