Edited Azin1 cooperates with Ddx1 to regulate hematopoiesis. (A) Colony formation of c-Kit⁺ BM cells after transduction with Ddx1 knockdown (KD) vectors. Colonies were counted in the different groups after 7 days of incubation, including the mix, GM, and E groups (n = 5 per group; 3 independent experiments; Error bars represent SD; NC, negative control). (B) The percentage of early and late apoptotic c-Kit⁺ BM cells after transduction with Ddx1 knockdown vectors (n = 3 per group; 3 independent experiments; Error bars represent SEM from 3 replicates). (C) The co-varying genes in the context of Ddx1 knockdown and edited Azin1 overexpression. The most prevalent 444 genes that decreased with Ddx1 knockdown but increased with Azin1-G overexpression are indicated in red. (D) Colony-forming ability of transduced cells. Colonies in the different groups were counted after 7 days of incubation. C omparison between the Azin1-G and Azin1-TC groups is shown in Figure 4B (n = 5 per group; two independent experiments; Error bars represent SD). (E) The change in genomic distribution of DDX1 after nonedited (Azin1-TC) or edited (Azin1-G) overexpression. (F) The expression of hematopoietic-related genes bound by DDX1 selectively in the edited Azin1 group. (G) The extent of DDX1 binding on Plaur and Tlr2 in the edited (Azin1-G) vs the nonedited (Azin1-TC) group. (H) AZI ChIP-quantitative polymerase chain reaction on the target gene loci in Azin1-G/Azin1-TC–overexpressing cells (n = 3 per group; 2 independent experiments; Error bars represent SD). (I) Colony forming ability of transduced cells. c-Kit⁺ BM cells were transduced with GFP-tagged edited (Azin1-G) or nonedited (Azin1-TC) overexpression lentiviruses; 48 hours after transduction, GFP⁺ cells were sorted and transduced with mCherry-tagged Plaur knockdown lentivirus. GFP⁺ mCherry⁺ cells were sorted for CFU assay 48 hours after transduction with Plaur knockdown lentivirus. Depletion of Plaur had a stronger effect on edited Azin1 (Azin1-G) than unedited Azin1 (Azin1-TC) cells (2.7-fold vs 1.5-fold decrease) (n = 5 per group; 2 independent experiments; Error bars represent SD). (J) Colony-forming ability of transduced cells. FigureCompared with the Empty-Ctrl, the Plaur-overexpressing cells generated more colonies. Overexpression of Plaur in the fully edited Azin1 (Azin1-G) and nonedited Azin1 (Azin1-TC) cells increased their colony-forming abilities. Overexpression of Plaur rescued the colony-forming abilities of nonedited Azin1 c-Kit⁺ cells, making these comparable to the edited group (n = 5 per group; 3 independent experiments; Error bars represent SD). (K) Schematic showing how AZI might interact with DDX1 to regulate hematopoiesis. Edited AZI is translocated from the cytoplasm to the nucleus where it interacts with DDX1 with high affinity to increase DDX1 binding on hematopoietic-regulated genes. DDX1 then activates target gene expression and sustains normal HSC differentiation. However, nonedited AZI preferentially localizes to the cytoplasm, forcing HSCs to proliferate and leading to failed HSC reconstitution in recipients. *P < .05; **P < .01; ***P < .001.