Figure 1. Targeting Ubiquitin to Cas9 to Increase Its Degradation.

(A) Schematic of the Ubiquitin-Cas9 (Ubi-Cas9) vector for the expression of Cas9 with short half-life. Red box indicates the inserted ubiquitin-targeting signal that is tagged to the N-terminus of Cas9. Green box indicates T7 promoter. The C-terminus of Cas9 carries the nuclear localization signal (NLS). (B) Western blot analysis for comparing the half-life of WT-Cas9 and Ubi-Cas9 in transfected HEK293 cells that were treated with 50 μM cycloheximide for different times (0, 4, 8, 12, 24 h) to inhibit protein synthesis. The relative protein levels of WT-Cas9 and Ubi-Cas9 were assessed by densitometric analysis of their bands on western blots, and the value at 0 h was considered 100% (mean ± SEM; n = 3, *p < 0.05; **p < 0.01). Images of full-length gels are seen in supplemental information. (C) Immunocytochemical assays showing the rapid degradation of Ubi-Cas9 in monkey embryos after injection of Ubi-Cas9 mRNA for 24 h or 40 h. WT-Cas9 mRNA injection served as controls. Embryos were stained with anti-Cas9 (green) and DAPI (blue) for nuclear staining. Note that more Ubi-Cas9 is located in the nucleus than the cytoplasm. Scale bar: 20 μm.