Dnmt3a loss increases self-renewal capacities in Jak2VFHSCs in vitro. (A) Experimental design, WT, and Jak2VF LSKs were sorted, transduced with lentivirus coding Cas9 coupled to a GFP reporter plus or minus sg-RNA targeting Dnmt3a. Thirty-six hours following transduction, 10k cells were seeded into methyl cellulose plates in triplicate; CFUs were counted and seeded weekly. (B) CFU count upon serial replating (4weeks) of LSKs derived cells from (left to right) WT, WT-Cas9, WT-Dnmt3a-Cas9, Jak2VF, Jak2VF-Cas9, Jak2VF-Dnmt3a-Cas9 conditions. (C) GFP expression upon cells replating (percentage) in WT-Cas9, WT-Dnmt3a-Cas9, Jak2VF-Cas9, Jak2VF-Dnmt3a-Cas9 LSKs derived cells. (D) Sequence alignment of the Dnmt3a gene at sgRNA binding loci within Jak2VF-Cas9 at week 1 (p1) and Jak2VF-Dnmt3a-Cas9 LSK-derived cells at week 5 (p5). (E) Dnmt3a transcription related peaks showing gene expression (read intensity) in Jak2VF-Cas9 (p1) and in Jak2VF-Dnmt3a-Cas9 (P5) at Dnmt3a-Cas9 target loci. (F) Principal component analysis (PCA) plot showing gene-expression profile of the LSK-derived cells from Jak2VF-Cas9 -p1 (black), Jak2VF-Dnmt3a-Cas9-p1 (orange), Jak2VF-Dnmt3a-Cas9-p5 (dark blue). Each dot represents an independent biological sample. (G) Categorized gene enriched in Jak2VF-Cas9 and Jak2VF-Dnmt3a-Cas9 gene expression (RNA-Seq). Gene-set enrichment of transcriptional pathways link to cell cycle, breast cancer, stemness, DNA damage, and hypomethylation-mediated gene expression.