Figure 4: Construct optimization enhances inducible activation of split CRISPR.
From: Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity
(a) Comparison of split CRISPRa-mediated activity against two previously published systems24,27. The VP64 domain in a recently reported rapamycin-inducible system (pX855/pX856)24 was replaced with the VPR domain to create pX-VPR. The 4-OHT-inducible intein domain27 was inserted into the K218/S219 site of the full-length dCas9-VPR construct, which also contains the S219C mutation for protein splicing, to create intein-VPR. The luciferase reporter cells were transfected with plasmids expressing either ERT-A-VPR, ERT-C-VPR, pX-VPR or intein-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. (b) Surveyor assay of indels mediated by the optimized inducible split Cas9, ERT-C, at PHOX2B gene locus. Indel rates are presented as means±standard deviation for three biological replicates. ND, not detected. Asterisk (*) denotes a non-specifically amplified DNA band that is excluded from indel analyses. (c) Luciferase activity of reporter cells expressing ERT-C-VPR or ERT-C*-VPR, which does not contain the FRB/FKBP domains (n=3 biological replicates). The luciferase activity is presented as mean±standard deviation for three biological replicates. (d) An inducible split CRISPR system derived from Staphylococcus areus Cas9 variant25. One sgRNA (sg-Sa-POU5F1) was expressed to target upstream of the transcription start site. Transcriptional activation of endogenous Oct4 expression was mediated by ERT-Sa-VPR in HEK293T cells induced with 4OHT (10 μM) and/or Rap (10 nM). The mRNA expression levels were measured by RT–qPCR for three biological replicates 48 h after induction. Fold-increase values are presented as mean±standard deviation relative to a negative control sgRNA. (e) Comparison in luciferase activity between cells expressing ERT-A-VPR and GR-A-VPR (n=3 biological replicates). The luciferase reporter cells expressing either ERT-A-VPR or GR-A-VPR were induced with either 4OHT (10 μM) or Dex (dexamethasone, 10 μM), and/or Rap (10 nM). The normalized activities are presented as mean±standard deviation.
