FIG 3.

Disruption of ATF4-binding motif in +55 enhancer of BCL11A is an attractive alternative therapeutic approach. (A) Schematic representation of ATF4-binding motif in the +55 enhancer of BCL11A with indication of SpyCas9 and enAspCas12a target sites. (B) Quantification of editing efficiency at 3 days post-electroporation by SpyCas9 RNPs programmed with Spy1617 or SpyTS1 sgRNA, or enAspCas12a programmed with EnAspTS2 crRNA in CD34+ HSPCs from six donors. (C) Quantification of HbF induction in differentiated erythrocytes (18 days post-electroporation) CD34+ HSPCs from six donors treated with SpyCas9 RNPs programmed with Spy1617 or SpyTS1, or enAspCas12a programmed with EnAspTS2 crRNA. (D) Quantification of editing efficiency at 3 days post-electroporation by 1xNLS enAspCas12a-HF1 and 3xNLS enAspCas12a-HF1 at EnAspTS2 in CD34+ HSPCs from four donors. (E) Quantification of HbF induction in differentiated erythrocytes (18 days post-electroporation) disrupted in CD34+ HSPCs from four donors with 1xNLS enAspCas12a-HF1 and 3xNLS enAspCas12a-HF1 at EnAspTS2. HF1 denotes the high-fidelity version of the Cas12a nuclease. Each donor is represented by a specific-colored shape as noted in the legend. Dashed lines indicated the grand mean of the group. Results were obtained from six and four independent experiments and presented as mean ± SEM. ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test between 1xNLS and 3xNLS enAspCas12a.