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. 2022 Jun 3;8(22):eabm9106. doi: 10.1126/sciadv.abm9106

Fig. 2. Spacer-nick–based gene correction of mutation hotspots at the HBB, ELANE, IL7R, and PRF1 loci in human HSPCs.

Fig. 2.

(A) Strategy to repair mutations occurring in the exons 1 and 2 of HBB, in the exons 4 and 5 of ELANE, or in coding and intronic sequences of IL7R and PRF1 genes. A pair of spacer-nick sgRNAs targeting the HBB, ELANE, IL7R, and PRF1 loci; and their spacer distances are indicated. Donor templates with indicated length of HAs including repair sequence and a Sal I recognition site in the cases of HBB and ELANE or codon-modified cDNA and poly-A sequences in the cases of IL7R and PRF1 are depicted. Primers used to amplify the targeted alleles are shown. (B) Pie charts showing percentages of WT (gray), NHEJ (blue), and HDR (orange) sequences at the targeted HBB, ELANE, IL7R, and PRF1 loci in the HSPCs treated with either sgRNAs/Cas9 (Cas9) or spacer-nick (Cas9n) RNPs and AAV donor vectors. The number of analyzed reads is indicated in center of the pie charts. Data are represented from three independent experiments. (C) Bar graphs showing the ratio of HDR:NHEJ events shown in (B) at the indicated loci in human HSPCs. Data are shown as means ± SD and based on three independent experiments. ***P < 0.001; **P < 0.01 (Mann-Whitney test).