Figure 6.

Investigating Gene-Editing Outcomes at Euchromatin versus Heterochromatin Using Cell Cycle-Regulated Cas9

(A) Constitutive and cell cycle-dependent post-translational regulation of Cas9 activity. Regular Cas9 is stable throughout the cell cycle, inducing DSBs at stages in which HDR is either not active or has to compete with NHEJ; Cas9^hGem(1/110), in contrast, owing to APC/C-Cdh1-mediated ubiquitination (Ub) and subsequent proteolysis at the M-to-G1 transition, preferentially accumulates during the HDR-permissive S-G2 phases. (B) Schematics of the main components of Cas9 and Cas9^hGem(1/110) expression constructs. Orange box with broken arrow, chimeric regulatory elements including the human cytomegalovirus immediate-early enhancer and the chicken β-actin promoter; orange box with vertical arrowhead, bovine growth hormone polyadenylation signal; yellow boxes, nuclear localization signals; black oval, Cas9 ORF; red oval, DNA coding for the first 110 residues of human Geminin, hGem(1/110). (C) Dual-color flow cytometric quantification of HDR and NHEJ events at euchromatin versus heterochromatin using Cas9 or Cas9^hGem(1/110). HER.TLR^TetO.KRAB cells, incubated or not incubated with Dox, were exposed to Plasmid^d and the indicated gRNAs together with Cas9 (open bars) or Cas9^hGem(1/110) (solid bars). (D) Dual-color flow cytometric quantification of HDR and NHEJ events induced by Cas9 or Cas9^hGem(1/110) in control HER.TLR^KRAB cells. HER.TLR^KRAB cells, treated or not treated with Dox, were exposed to Plasmid^d and the indicated gRNAs together with Cas9 (open bars) or Cas9^hGem(1/110) (solid bars). (E) Relative participation of HDR and NHEJ pathways during plasmid-mediated repair of DSBs created at heterochromatin versus euchromatin. Net result of the data shown in (C) corresponds to the ratios between the frequencies of NHEJ and HDR in HER.TLR^TetO.KRAB cells exposed or not exposed to Dox. Bars represent mean ± SD of three independent experiments (biological replicates done on different days). The p values varied from a minimum of 5 × 10⁻⁴ to a maximum of 8.5 × 10⁻³; p < 0.05 was considered significant. (F) Relative participation of HDR and NHEJ pathways during plasmid-mediated repair of DSBs in control HER.TLR^KRAB cells. Net result of the data shown in (D) corresponds to the ratios between the frequencies of NHEJ and HDR in HER.TLR^KRAB cells incubated or not incubated with Dox. Bars represent mean ± SD of three independent experiments (biological replicates done on different days). The p values varied from a minimum of <1 × 10⁻⁴ to a maximum of 3.7 × 10⁻²; p < 0.05 was considered significant.