Figure 3: Tight control of inducible split Cas9 with ligand-binding domain ERT.

(a) Inducible split dCas9 was optimized by fusing ERT to each split fragment to create a split dCas9 responsive to rapamycin (Rap) and 4-hydroxytamoxifen (4OHT). In the N-terminal fragment, the ERT domain is fused to the C-terminus following the FRB domain. Vice versa, the ERT domain is fused to the N-terminus of the C-terminal fragment followed by the FKBP domain. The activation domain used for CRISPRa is either VP64 or VPR domain. The luciferase reporter cells were transfected with plasmids expressing either ERT-A-VP64 or ERT-A-VPR, and induced with 4OHT (10 μM) and/or Rap (10 nM). The data are displayed as mean±standard deviation of the luciferase activity for three biological replicates. pCBh, chicken β-actin promoter. (b) Luciferase activity of cells expressing ERT-A-VPR measured 2, 4, 8, 16, 24 and 48 h after induction with 4OHT (10 μM) and/or Rap (10 nM; n=3 biological replicates for each time point). Values are normalized to a positive control, which is the wild-type dCas9-VPR with an sgRNA-targeting GAL4 sequence, and background-subtracted from a negative control, which is wild-type dCas9-VPR with a negative sgRNA. The data are presented as mean±standard deviation. (c) The optimized inducible split ERT-A-VPR facilitates tunable transcriptional activation of reporter gene. The luciferase reporter cells were transfected with plasmids expressing ERT-A-VPR, and induced with various concentrations of 4OHT and Rap (n=3 biological replicates). The data are shown as mean±standard deviation. (d) ERT-A-VPR mediates inducible transcriptional activation of endogenous Oct4 expression in HEK293T cells, measured by RT–qPCR. Two previously validated sgRNAs (sgPOU5F1-3 and sgPOU5F1-5) were expressed to target upstream of the transcription start site¹⁹. Cells were induced with 4OHT (10 μM) and/or Rap (10 nM). Fold-increase values are mRNA expression levels (mean±standard deviation) relative to a negative control sgRNA.