Comparison of prime- and base-editing inducing HPFH/HPFH-like mutations in SCD HSPCs and their erythroid progeny. (A) Schematic representation of the β-globin locus on chromosome (chr) 11 including the HBG1 and HBG2 genes and their promoters (prom). The light and dark gray bars denote the gRNAs used with BEs and their PAM, respectively6,13,17-21 (supplemental Table 1). In the –200 region, gKLF198 induces the T>C (A>G) HPFH mutation at the –198 site generating a KLF1 motif, with a bystander at position –199. gTAL175-NG and gTAL175 generate a TAL1 motif by introducing the HPFH T>C (A>G) mutation at the –175 site, using NG (dotted line) and NGG (solid line) PAMs, respectively. In the –115 region, gKLF123/124 induces T>C (A>G) mutations at the –123 and –124 sites, creating a KLF1 BS. gBCL113 creates an A>G (T>C) mutation at the –113 site, generating GATA1 BS and A>G mutation at the –116 site disrupting the BCL11A BS and bystander edits at the –112 and/or –110 sites. (B) Experimental protocol used for prime and base editing experiments in peripheral blood (donor 1) or bone marrow (donors 2 and 3) CD34+ HSPCs from SCD donors. HSPCs were transfected with PEs using the protocol described in the legend to Figure 2B (supplemental Table 1). For base editing, cells were transfected with 3 μg of in vitro-transcribed BE mRNA (as previously described6) and 1 or 2 guide RNAs (100 pmol each; Integrated DNA Technologies; supplemental Table 1). Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. After transfection, bulk HSPCs (ie, without any selection) were either cultured in preactivation medium or differentiated in mature RBCs following the previously described method6,14 or plated in a semisolid medium to induce erythroid differentiation (colony-forming assay). The editing efficiency was evaluated in bulk HSPCs and erythroid precursors 6 days posttransfection and in single BFU-Es by NGS (Illumina, as previously described6 using the primers and probes described in supplemental Table 2). The presence of the 4.9-kb deletion was evaluated by ddPCR and PCR as described in the Figure 2 legend. PE and BE efficiency was evaluated following the method described in the Figure 2B legend and the CRISPResso2 webtool,22 respectively. Globin mRNA expression was measured in erythroid precursors and single BFU-Es using qRT-PCR. Hemoglobin, erythroid markers, and enucleation were analyzed by flow cytometry in erythroid precursors and mature RBCs. Sickling assay was performed on mature RBCs. (C-D) Percentage of NGS reads in the HBG promoters in (C) HSPCs and (D) erythroid precursors. Prime editing modifications were reported as indicated in Figure 2C. Base editing was performed using the gRNAs displayed in panel A and ABE8-13m or ABEmax-NG. In the –115 region, +G, –B, and +K refer to GATA1 BS insertion (–113), BCL11A BS (–116) disruption, and KLF1 BS insertion (–124/123), respectively. In the –200 region, +K and +T create the KLF1 (–198) and TAL1 (–175) BSs, respectively. Bystanders (Byst.) were found in positions –110, –112, and –199. Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. (E) NGS reads from the HSPCs of donor 3, ABE8-13m, and gBCL113 generate a new GATA1 motif (blue) and disrupts the BCL11A BS (red). Editing with gKLF123/124 leads to the generation of a KLF1 motif (orange). Multiplex base editing using both gRNAs showed the presence of dual editing events (dual edit) and a high rate of InDels, as shown in the nucleotide distribution around the gRNAs. At each base in the reference amplicon, the percentage of each base as observed in sequencing reads is shown (A = green, C = orange, G = yellow, and T = purple). Black bars show the percentage of reads for which that base was deleted. Brown bars between bases show the percentage of reads having an insertion at that position (top). ABE8-13m and gKLF198 create a KLF1 motif (green). Editing with ABE8-13m and gTAL175 induces the generation of a TAL1 motif (pink). Both gRNAs together allow dual editing (bottom). (F) Frequency (%) of the 4.9-kb deletion measured by ddPCR in HSPCs and erythroid precursors from 3 donors with SCD following editing with PEs or BEs. Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. The gray area indicates background 4.9-kb deletion levels (ie, observed in the control-treated samples). (G) Agarose gel image showing a band corresponding to the PCR product (HBG1/2 junction) obtained using primers surrounding the HBG1/2 junction (generated upon deletion of the 4.9-kb region) in control (ctrl; mock-transfected or transfected with the PEnmax or ABE8-13m mRNA alone) SCD HSPCs or SCD erythroid precursors and treated with PE or BEs (donor 3). For the positive control, we used DNA from K562 cells displaying 60% of 4.9-kb deletion measured by ddPCR (ctrl+). For the negative control, we performed the PCR reaction in the absence of DNA (ctrl–). (H) Frequency of CD36+, CD71+, CD235a+, and enucleated RBCs at day 13 (light gray) and 20 (dark gray) of erythroid differentiation, as measured by flow cytometry analysis of CD36, CD71, and CD235a erythroid markers and by DRAQ5 staining for donors 1 and 2. Control (ctrl) samples were mock-transfected. (I) Globin mRNA expression in erythroid precursors from 3 donors was measured by qRT-PCR. We reported the editing frequency below each graph. Control (ctrl) samples were mock-transfected. (J) Flow cytometry histograms showing the frequency of HbF+ cells in erythroid precursors from donors 1 and 2. For each sample, we also reported the editing frequency. Control (ctrl) samples were mock-transfected. (K) Frequency of sickling RBCs after 2-hour incubation under hypoxic conditions (0% O2) for donor 1. We reported the editing frequency below each graph. Control (ctrl) samples were mock-transfected. (L) Globin mRNA expression in single BFU-Es from donor 1 was measured by qRT-PCR. We reported the editing frequency below each graph. Control (ctrl) samples were transfected only with the PEnmax mRNA. Statistical significance was assessed using an unpaired Kruskal-Wallis test with multiple comparison (Dunn’s correction) and displayed as ∗∗∗∗P < .001, ∗∗P < .01, and ∗P < .05.

Comparison of prime- and base-editing inducing HPFH/HPFH-like mutations in SCD HSPCs and their erythroid progeny. (A) Schematic representation of the β-globin locus on chromosome (chr) 11 including the HBG1 and HBG2 genes and their promoters (prom). The light and dark gray bars denote the gRNAs used with BEs and their PAM, respectively6,13,17-21 (supplemental Table 1). In the –200 region, gKLF198 induces the T>C (A>G) HPFH mutation at the –198 site generating a KLF1 motif, with a bystander at position –199. gTAL175-NG and gTAL175 generate a TAL1 motif by introducing the HPFH T>C (A>G) mutation at the –175 site, using NG (dotted line) and NGG (solid line) PAMs, respectively. In the –115 region, gKLF123/124 induces T>C (A>G) mutations at the –123 and –124 sites, creating a KLF1 BS. gBCL113 creates an A>G (T>C) mutation at the –113 site, generating GATA1 BS and A>G mutation at the –116 site disrupting the BCL11A BS and bystander edits at the –112 and/or –110 sites. (B) Experimental protocol used for prime and base editing experiments in peripheral blood (donor 1) or bone marrow (donors 2 and 3) CD34+ HSPCs from SCD donors. HSPCs were transfected with PEs using the protocol described in the legend to Figure 2B (supplemental Table 1). For base editing, cells were transfected with 3 μg of in vitro-transcribed BE mRNA (as previously described6) and 1 or 2 guide RNAs (100 pmol each; Integrated DNA Technologies; supplemental Table 1). Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. After transfection, bulk HSPCs (ie, without any selection) were either cultured in preactivation medium or differentiated in mature RBCs following the previously described method6,14 or plated in a semisolid medium to induce erythroid differentiation (colony-forming assay). The editing efficiency was evaluated in bulk HSPCs and erythroid precursors 6 days posttransfection and in single BFU-Es by NGS (Illumina, as previously described6 using the primers and probes described in supplemental Table 2). The presence of the 4.9-kb deletion was evaluated by ddPCR and PCR as described in the Figure 2 legend. PE and BE efficiency was evaluated following the method described in the Figure 2B legend and the CRISPResso2 webtool,22 respectively. Globin mRNA expression was measured in erythroid precursors and single BFU-Es using qRT-PCR. Hemoglobin, erythroid markers, and enucleation were analyzed by flow cytometry in erythroid precursors and mature RBCs. Sickling assay was performed on mature RBCs. (C-D) Percentage of NGS reads in the HBG promoters in (C) HSPCs and (D) erythroid precursors. Prime editing modifications were reported as indicated in Figure 2C. Base editing was performed using the gRNAs displayed in panel A and ABE8-13m or ABEmax-NG. In the –115 region, +G, –B, and +K refer to GATA1 BS insertion (–113), BCL11A BS (–116) disruption, and KLF1 BS insertion (–124/123), respectively. In the –200 region, +K and +T create the KLF1 (–198) and TAL1 (–175) BSs, respectively. Bystanders (Byst.) were found in positions –110, –112, and –199. Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. (E) NGS reads from the HSPCs of donor 3, ABE8-13m, and gBCL113 generate a new GATA1 motif (blue) and disrupts the BCL11A BS (red). Editing with gKLF123/124 leads to the generation of a KLF1 motif (orange). Multiplex base editing using both gRNAs showed the presence of dual editing events (dual edit) and a high rate of InDels, as shown in the nucleotide distribution around the gRNAs. At each base in the reference amplicon, the percentage of each base as observed in sequencing reads is shown (A = green, C = orange, G = yellow, and T = purple). Black bars show the percentage of reads for which that base was deleted. Brown bars between bases show the percentage of reads having an insertion at that position (top). ABE8-13m and gKLF198 create a KLF1 motif (green). Editing with ABE8-13m and gTAL175 induces the generation of a TAL1 motif (pink). Both gRNAs together allow dual editing (bottom). (F) Frequency (%) of the 4.9-kb deletion measured by ddPCR in HSPCs and erythroid precursors from 3 donors with SCD following editing with PEs or BEs. Control (ctrl) samples were either mock-transfected or transfected with the PEnmax or the ABE8-13m mRNA alone. The gray area indicates background 4.9-kb deletion levels (ie, observed in the control-treated samples). (G) Agarose gel image showing a band corresponding to the PCR product (HBG1/2 junction) obtained using primers surrounding the HBG1/2 junction (generated upon deletion of the 4.9-kb region) in control (ctrl; mock-transfected or transfected with the PEnmax or ABE8-13m mRNA alone) SCD HSPCs or SCD erythroid precursors and treated with PE or BEs (donor 3). For the positive control, we used DNA from K562 cells displaying 60% of 4.9-kb deletion measured by ddPCR (ctrl+). For the negative control, we performed the PCR reaction in the absence of DNA (ctrl–). (H) Frequency of CD36+, CD71+, CD235a+, and enucleated RBCs at day 13 (light gray) and 20 (dark gray) of erythroid differentiation, as measured by flow cytometry analysis of CD36, CD71, and CD235a erythroid markers and by DRAQ5 staining for donors 1 and 2. Control (ctrl) samples were mock-transfected. (I) Globin mRNA expression in erythroid precursors from 3 donors was measured by qRT-PCR. We reported the editing frequency below each graph. Control (ctrl) samples were mock-transfected. (J) Flow cytometry histograms showing the frequency of HbF+ cells in erythroid precursors from donors 1 and 2. For each sample, we also reported the editing frequency. Control (ctrl) samples were mock-transfected. (K) Frequency of sickling RBCs after 2-hour incubation under hypoxic conditions (0% O2) for donor 1. We reported the editing frequency below each graph. Control (ctrl) samples were mock-transfected. (L) Globin mRNA expression in single BFU-Es from donor 1 was measured by qRT-PCR. We reported the editing frequency below each graph. Control (ctrl) samples were transfected only with the PEnmax mRNA. Statistical significance was assessed using an unpaired Kruskal-Wallis test with multiple comparison (Dunn’s correction) and displayed as ∗∗∗∗P < .001, ∗∗P < .01, and ∗P < .05.

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