Fig. 2: Optimization of prime editing HBB in human CD34+ HSPCs from healthy donors and from SCD patients.
Healthy donor HSPCs (5 × 105) and SCD patient HPSCs (4–5 × 106) were electroporated. After electroporation, all healthy HSPCs and 1 × 105 SCD patient HSPCs were cultured for 3 and 6 d before genomic DNA extraction. The remaining SCD patient HSPCs were cryopreserved for later mouse engraftment experiments. Bars reflect mean ± s.d. with replicate values shown as individual dots. For healthy donor HSPC editing, n = 3 independent biological replicates from three different donors. For SCD patient donor editing, n = 4 independent biological replicates from four different donors. All editing values include only the desired prime editing product with no indels or other changes at the target site. Indels are shown as separate bars in each plot. a, Quantification of editing efficiencies for different nicking sgRNAs targeting HBB. b, Distance between the epegRNA-induced nick and the nicking sgRNA-induced nick on the opposite strand for four nicking sgRNA candidates at HBB. In the PE3b strategy, nicking of the unedited strand cannot take place until after editing and ligation of the other strand is complete. c, Components and total combined volume of PEmax mRNA, epegRNA and nicking sgRNAs for various PE3max electroporation strategies. d, Editing efficiency quantification for each condition listed in c. e, Quantification of editing efficiencies using the 2xPE3max strategy with the top-performing nicking sgRNAs from a. f, Editing efficiency of reversion of the pathogenic HBBS allele back to HBBA in SCD patient CD34+ HSPCs with 2xPE3max + NG1. A total of 4–5 × 106 cells were edited per donor in parallel electroporations of 5 × 105 cells per replicate and pooled together for subsequent in vitro culture or cryopreserved for later injection into mice.
