Figure 2.
Functional analyses of in situ gene-edited HSCs. (A) Frequency of tdTomato+ cells among HSCs (CD150+CD48−LSK) or among the indicated subsets of BM progenitors in adult injected animals 5 weeks after receiving 5 × 1012 vg per animal scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. Data compiled from 4 independent experiments. Females, square; males, triangles. (B) Frequency of primitive progenitors within tdTomato− and tdTomato+ subsets of LinloKit+ BM progenitors at 5 weeks after injection of adult animals with 5 × 1012 vg per animal scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 4 independent experiments. (C) Experimental design. For CFU assay, HSCs from animals injected with vehicle or NT vector (all tdTomato−), and tdTomato+ or tdTomato− HSCs from animals receiving 5 × 1012 vg per animal scAAV carrying targeting gRNAs, were sorted 5 weeks after injection and plated in methylcellulose. Colonies were scored visually at day 12. For transplant, LinloKit+ HSPCs from the tdTomato+ or tdTomato− fractions of the BM were sorted and transplanted into sublethally irradiated NSG recipients. Peripheral blood was analyzed by FACS every 4 weeks, and mice were euthanized >1 week after last bleed for BM chimerism analysis (Table 1; supplemental Figure 6 for results). (D) Percentages of CFU-GEMM, BFU, and CFU-GM formed from HSCs sorted from animals injected with vehicle or NT vector, or from tdTomato+ or tdTomato− HSCs sorted from animals injected as adults with 5 × 1012 vg per animal scAAV carrying targeting gRNAs. Total number of colonies scored is as follows: vehicle, 288; NT vector, 264; tdTomato−, 279; and tdTomato+, 225. Individual colony numbers provided in supplemental Table 2. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 2 independent experiments. BFU-E, burst-forming unit-erythroid; CFU, colony-forming unit; GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte; GM, granulocyte, macrophage; NT, nontargeting. Panel C created with biorender.com. Karimzadehfard A. (2025) https://biorender.com/mz1lx3u.

Functional analyses of in situ gene-edited HSCs. (A) Frequency of tdTomato+ cells among HSCs (CD150+CD48−LSK) or among the indicated subsets of BM progenitors in adult injected animals 5 weeks after receiving 5 × 1012 vg per animal scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. Data compiled from 4 independent experiments. Females, square; males, triangles. (B) Frequency of primitive progenitors within tdTomato− and tdTomato+ subsets of LinloKit+ BM progenitors at 5 weeks after injection of adult animals with 5 × 1012 vg per animal scAAV-2xgRNAs. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 4 independent experiments. (C) Experimental design. For CFU assay, HSCs from animals injected with vehicle or NT vector (all tdTomato−), and tdTomato+ or tdTomato− HSCs from animals receiving 5 × 1012 vg per animal scAAV carrying targeting gRNAs, were sorted 5 weeks after injection and plated in methylcellulose. Colonies were scored visually at day 12. For transplant, LinloKit+ HSPCs from the tdTomato+ or tdTomato− fractions of the BM were sorted and transplanted into sublethally irradiated NSG recipients. Peripheral blood was analyzed by FACS every 4 weeks, and mice were euthanized >1 week after last bleed for BM chimerism analysis (Table 1; supplemental Figure 6 for results). (D) Percentages of CFU-GEMM, BFU, and CFU-GM formed from HSCs sorted from animals injected with vehicle or NT vector, or from tdTomato+ or tdTomato− HSCs sorted from animals injected as adults with 5 × 1012 vg per animal scAAV carrying targeting gRNAs. Total number of colonies scored is as follows: vehicle, 288; NT vector, 264; tdTomato−, 279; and tdTomato+, 225. Individual colony numbers provided in supplemental Table 2. One-way ANOVA with Brown-Forsythe and Welch correction. ∗P < .05. Data compiled from 2 independent experiments. BFU-E, burst-forming unit-erythroid; CFU, colony-forming unit; GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte; GM, granulocyte, macrophage; NT, nontargeting. Panel C created with biorender.com. Karimzadehfard A. (2025) https://biorender.com/mz1lx3u.

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