Cas9 nickase editing results in seamless HBA2 deletion and reduced HSPCs toxicity. (A-B) HBA2 copies (A) and KI efficiency (B) in Cas9 (RNP) or Cas9 nickase (RNP D10A) edited HSPCs in erythroid liquid culture. Black lines represent mean. (C) Relative abundance of endogenous β and KI-β^AS3 mRNA at day 12 of erythroid liquid culture (mean ± SD, n = 3-4). (D) expression of β^AS3 transcripts normalized by copy number (CN) in Cas9- or Cas9 nickase-treated erythroblasts (mean ± SD, n = 4-5). (E) InDel quantification at gRNA target site in edited HSPCs. Black lines represent mean. (F) CFC number expressed as percentage of untreated control (UT). Bars represent mean ± SD (n = 3-4); red dashed line indicates 100%. (G) CFU frequency in edited HSPCs. BFU-E, burst-forming unit-erythroid; CFU-GM, CFU-granulocyte, macrophage. Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; Bars represent mean ± SD (n = 2-4). (H) Genotypes of BFU-E (n = 202) derived from RNP D10A + AAV HSPCs. Percentages are indicated. (I) Frequency of different InDel patterns in HBA2 deleted BFU-E treated with Cas9 (RNP, red; n = 28) or Cas9 nickase (RNP D10A, gray; n = 96). Editing was measured across HBA2 deletion junctions.