Impact of vector design on CRISPR-mediated gene editing of HSCs and more mature hematopoietic lineages in situ in adult Ai9;SpCas9-EGFP mice. (A) Schematic of Ai9 transgene in Rosa26 locus. Double-stranded break induction and excision of LoxP-flanked STOP cassette can be detected by robust tdTomato expression. Triangles: LoxP sites. (B) Vector design. ssAAV or scAAVs carrying gRNAs driven by U6, H1, or 7SK promoters. ssAAV-1xgRNAs and scAAV-1xgRNAs carry 1 copy of each gRNA, whereas scAAV-2xgRNAs encode 2 copies of each gRNA. (C-E) Frequency of tdTomato⁺ cells among BM HSCs in adult (5 to 8-week-old) animals receiving IV injections of vehicle or AAV and analyzed 5 weeks after injection. Animals received vehicle only or 5 × 10¹¹ vg or 5 × 10¹² vg per animal of ssAAV-1xgRNAs (C), vehicle only or 5 × 10¹² vg of either ssAAV-1xgRNAs or scAAV-1xgRNAs (D), or vehicle only or 5 × 10¹² vg per animal of either scAAV-1xgRNAs or scAAV-2xgRNAs (E). One-way analysis of variance (ANOVA) with Brown-Forsythe and Welch correction. ∗P < .05; ∗∗∗P < .001. Data compiled from 2 (panels C-D) or 4 (panel E) independent experiments. Females, square; males, triangles. (F) Frequency of tdTomato⁺ cells among different cellular subsets in the spleen, BM, or thymus of Ai9;SpCas9-EGFP mice injected with vehicle or 5 × 10¹² vg per mouse scAAV-2xgRNAs. B cell, B220⁺; T cell, CD3⁺; myeloid, B220⁻CD3⁻, EryA, CD71^highTer119⁺; EryB, CD71^intermediateTer119⁺, EryC, CD71⁻Ter119⁺; CD4 SP, CD3⁺CD4⁺CD8⁻; DP, CD3⁺CD4⁺CD8⁺; DN, CD3⁺CD4⁻CD8⁻; CD8 SP, CD3⁺CD4⁻CD8⁺. Unpaired t test with Welch correction. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data compiled from 3 independent experiments. dITR, defective inverted terminal repeat; DN, double negative; DP, double positive; ITR, inverted terminal repeat; EryA, basophilic erythroblasts; SP, single positive. Panels A and B created with biorender.com. Karimzadehfard A. (2025) https://biorender.com/1fldmz.