We performed CRISPR KO screens in 3-week expanded primary mouse HSCs by targeting ~7,000 genes in the functional categories of transcription factors, kinases, phosphatases, known drug targets and genes associated with apoptosis and cancer. To target these ~7,000 genes, we used a 10 sgRNA/gene sgRNA library with ~70,000 gene targeting sgRNAs and ~5,000 control sgRNAs. We transplanted ~17x106 day-14 CRISPR library cells into 17 lethally irradiated mice, which resulted in high level multi-lineage donor peripheral blood chimerism. After 10-weeks, bone marrow and spleen from these mice were collected and pooled before various hematopoietic cell types purified for sgRNA sequencing: c-Kit+Sca1+Lineage- HSCs, Mac1/Gr1+ myeloid cells, CD4/CD8+ T cells, B220+ B cells, and Ter119+ erythroid cells. This enabled four pairwise screen comparisons of various HSC-derived blood cells for putative regulators of hematopoiesis revealing genetic regulators of the lineage trajectory from HSCs to myeloid cells, T-cells, B-cells and erythroid cells.
See Haney et. al. 2022 bioRxiv for more details.