Inversion of intervening sequence can also reactivate silenced genes. (A) A schematic representation of the HBB locus on chromosome 11 depicting the hypersensitive sites of the LCR (light blue), HBE (brown), HBG2 (pink), HBG1 (green), HBD (light purple), and HBB (dark purple) genes. Below (left) is a zoom-in on HS2 and HS3 showing the position of gRNA-HS2/3. On the right side, a zoom-in on the HBG2 promoter with the most relevant TF-BS (gray) and position of gRNA_500. At the bottom, the 27-kb deletion generated by the combination of gRNA_500 and gRNA-HS2/3. Below, a representation of the inversion generated by the same gRNAs. (B) RT-qPCR analysis of HBB, HBG1, and HBG2 mRNA levels in proliferating bulk cell populations. HBB, HBG1, and HBG2 mRNA expression was normalized to actin and is displayed as the percentage of HBB + HBG1 + HBG2. WT is the parental lentiviral Cas9-expressing cell line without gRNAs. (C) Flow cytometry plots showing the percentage of HbF-positive cells in proliferating bulk cell populations. (D) On the left side, RT-qPCR analysis of HBB, HBG1, and HBG2 mRNA levels in 2 proliferating heterozygous deletion clones. HBB, HBG1, and HBG2 mRNA expression was normalized to actin and displayed as a percentage of HBB + HBG1 + HBG2. On the right side, HbF of 2 clonal cell lines as measured by HPLC. Percentage of HbF calculated over the total Hb tetramers. (E) Same as panel D but for 3 HS2/3-500 inversion clones. (F) Same as panel A but a schematic representation of the gRNAs HS2 and HBG1/2, cutting between HBG1 and HBG2. (G) RT-qPCR analysis of the HBB, HBG1, and HBG2 mRNA levels in proliferating bulk cell populations. HBB, HBG1, and HBG2 mRNA expression was normalized to actin and displayed as a percentage of HBB + HBG1 + HBG2. (H) Flow cytometry plot showing the percentage of HbF-positive cells in proliferating bulk cell population.