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. 2021 Sep 29;10:e70557. doi: 10.7554/eLife.70557

Figure 4. Deletion of 3′HS1 induces HbF in primary adult HSPC.

(A) The experimental scheme for primary HSPC editing. (B) The deletion of 3′HS1 and HS5 in three CD34+ peripheral blood mononuclear cell (PBMC) HSPCs from three individual adult donors. Refer to Figure 4—source data 1 for original gel picture. (C) The HbF+ cell percentage at day 21 in three HSPCs from three individual adult donors after 3′HS1 and HS5 deletion. p-Value is calculated by one-tailed paired t-test. n.s., not significant, p=0.3659 in HS5 deletion vs. Cas9 by one-tailed paired t-test. (D) The reprehensive flow plot for HbF+ cells at day 21 in 3′HS1-deleted and HS5-deleted PBMC HSPC. The data is from donor #1. (E) The model of fetal hemoglobin regulation through 3′HS1.

Figure 4—source data 1. The gel picture of paired guide deletion for HS5 and 3′HS1 in HSPC.

Figure 4.

Figure 4—figure supplement 1. 3’HS1 deletion in HSPC.

Figure 4—figure supplement 1.

(A) Giemsa–Wright staining of differentiated erythroid cells from HSPC electroporated with Cas9, 3′HS-1 guide RNA pair, and HS5 deletion guide RNA pair at differentiation culture day 21. Orange arrow indicates the enucleated red blood cells, and blue arrows indicate the reticulocytes. (B) Differentiation stage of three HSPCs by flow cytometry of band 3 from different donors electroporated with Cas9, 3′HS-1 guide RNA pair, and HS5 deletion guide RNA pair at differentiation culture day 21.