Figure 3.
Robust in situ HSC gene editing in neonatally injected mice. (A) Percentage TdTomato+ among BM HSCs at 8 weeks after injection of P1 to P3 neonates. Animals received vehicle control or the indicated dose (vg) of ssAAV-1xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 3 independent experiments. (B) Comparison of percentage tdTomato+ HSCs in animals receiving vehicle or 1 × 1012 vg per mouse of either ssAAV-1xgRNAs or scAAV-1xgRNAs or scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. Data compiled from 4 independent experiments. (C) Frequency of TdTomato+ cells among different cell lineages in the spleen, BM, and thymus of animals injected with vehicle or 5 × 1012 vg per animal scAAV-2xgRNAs. B cell, B220+; T cell, CD3+; myeloid, B220−CD3−; EryA, CD71highTer119+; EryB, CD71intermediateTer119; EryC, CD71−Ter119+; CD4 SP, CD3+CD4+CD8−; DP, CD3+CD4+CD8+; DN, CD3+CD4−CD8−; and CD8 SP, CD3+CD4−CD8+. Unpaired t test with Welch correction. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data compiled from 4 independent experiments. (D) Frequency of TdTomato+ cells among downstream BM progenitors in animals that received 1 × 1012 vg scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. Data compiled from 4 independent experiments. (E) Design of dual AAV vectors delivering separated CRISPR editing components. (F) Frequency of TdTomato+ HSCs in single or dual AAV delivery groups in neonatally injected Ai9;SpCas9-EGFP at 8 weeks after injection. Neonates were injected systemically with vehicle or with 2 × 1011 vg per animal of either scAAV-5′ gRNA only or scAAV-3′ gRNA only or scAAV-5′ gRNA and scAAV-3′ gRNA together or scAAV-2xgRNAs (ie, scAAV delivering 5′ gRNA and 3′ gRNA in 1 vector). One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 2 independent experiments. Females, square; males, triangles. dITR, defective inverted terminal repeat; ITR, inverted terminal repeat. Panel E created with biorender.com. Karimzadehfard A. (2025) https://biorender.com/3vjs8fl.

Robust in situ HSC gene editing in neonatally injected mice. (A) Percentage TdTomato+ among BM HSCs at 8 weeks after injection of P1 to P3 neonates. Animals received vehicle control or the indicated dose (vg) of ssAAV-1xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 3 independent experiments. (B) Comparison of percentage tdTomato+ HSCs in animals receiving vehicle or 1 × 1012 vg per mouse of either ssAAV-1xgRNAs or scAAV-1xgRNAs or scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. Data compiled from 4 independent experiments. (C) Frequency of TdTomato+ cells among different cell lineages in the spleen, BM, and thymus of animals injected with vehicle or 5 × 1012 vg per animal scAAV-2xgRNAs. B cell, B220+; T cell, CD3+; myeloid, B220−CD3−; EryA, CD71highTer119+; EryB, CD71intermediateTer119; EryC, CD71−Ter119+; CD4 SP, CD3+CD4+CD8−; DP, CD3+CD4+CD8+; DN, CD3+CD4−CD8−; and CD8 SP, CD3+CD4−CD8+. Unpaired t test with Welch correction. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data compiled from 4 independent experiments. (D) Frequency of TdTomato+ cells among downstream BM progenitors in animals that received 1 × 1012 vg scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. Data compiled from 4 independent experiments. (E) Design of dual AAV vectors delivering separated CRISPR editing components. (F) Frequency of TdTomato+ HSCs in single or dual AAV delivery groups in neonatally injected Ai9;SpCas9-EGFP at 8 weeks after injection. Neonates were injected systemically with vehicle or with 2 × 1011 vg per animal of either scAAV-5′ gRNA only or scAAV-3′ gRNA only or scAAV-5′ gRNA and scAAV-3′ gRNA together or scAAV-2xgRNAs (ie, scAAV delivering 5′ gRNA and 3′ gRNA in 1 vector). One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 2 independent experiments. Females, square; males, triangles. dITR, defective inverted terminal repeat; ITR, inverted terminal repeat. Panel E created with biorender.com. Karimzadehfard A. (2025) https://biorender.com/3vjs8fl.

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