Fig 1.
CjCas9 protein stability compared to SpCas9 protein.
A) Schematic representation of the size of SpCas9 and CjCas9. B) The HEK293T cells were transfected with the same number of copies of plasmids of either pX551-CMV-SpCas9 or of pX551-CMV-CjCas9. 72 h later, the numbers of plasmids inside the cells were determined by qPCR. C) CjCas9 and SpCas9 mRNAs were assessed by RT-qPCR with HPRT as the reference gene. D) The concentrations of the two Cas9 proteins fused with HA-tag were evaluated by western blot. Data are means ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t tests).
Fig 2.
The proteasome inhibitor MG132 increases CjCas9 protein level.
48 hours after transfection of the cells (in A HEK293T cells and in B HeLa cells) with the pX551-CMV-CjCas9-HA plasmid, the wells were treated with different doses of the proteasome inhibitor MG132 (1 to 5 μM) for 16 hours. The proteins were analyzed 3 days post transfection by western blot with an anti-HA antibody. β-actin was used as a loading control. Data are means ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t tests).
Fig 3.
Proteasome inhibitor bortezomib reduces CjCas9 degradation.
HEK293T cells (in A) and HeLa cells (in B) were transfected with equal concentrations of plasmids coding for HA-CjCas9 and treated with various concentrations of bortezomib (0, 6 and 12 nM) for 40 hours. Total cell extracts were analyzed by western blots with antibodies against an HA and β-actin (as a reference protein). Data are means ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t tests).
Fig 4.
Bortezomib increases the stability of CjCas9 synthesized under a weaker promoter than CMV.
The same number of plasmids coding for CjCas9-HA under different promoters, i.e., A) EFS or B) CBH, were transfected in HEK293T cells. Some of the wells was treated 18 hours after transfection with 12 nM of bortezomib for 40 hours. The cells were harvested and the level of the HA-CjCas9 protein was evaluated by western blots with β-actin as the reference protein. Untreated wells and CjCas9 under CMV promoter served as control. Data are mean ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t test).
Fig 5.
Bortezomib enhances the efficiency of CjCas9/ 2 sgRNA-mediated GAA repeat deletion in the FXN gene of HEK293T cells.
A) Schematic representation of the human FXN gene with the positions of the sgRNAs used in our experiment. B) Schematic description of the experiment: cells were co-treated with or without bortezomib (4 nM) and a plasmid encoding CjCas9 under CMV promoter and 2 sgRNAs under U6 promoters. C) The number of plasmid copies was determined by qPCR. D) The level of CjCas9 protein was quantified by western blot and normalized with β-actin. E) 72 hours after transfection, gene editing efficiency on FXN gene was measured by ddPCR. Data are means ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t tests).