Supplementary Figure 7 : Optimization of a A/C heterodimerizer drug-inducible dCas9-based activator.

From: Inducible and multiplex gene regulation using CRISPR–Cpf1-based transcription factors

Supplementary Figure 7

(a) Titrations of A/C heterodimerizer (left panel) and expression plasmids encoding dCas9-DmrA(x4) fusion protein and DmrC-VP64 or DmrC-p65 effectors (right panel) together with plasmids expressing either one or three sgRNAs targeted upstream of the human VEGFA transcription start site. Gene activation assessed by VEGFA ELISA; error bars represent s.e.m. for n = 3.

(b) Relative differences in gene activation between direct dCas9-activation domain fusions compared to drug-dependent dCas9-based activator fusions, using either one or three sgRNAs targeted upstream of the VEGFA transcription start site. Gene activation assessed by VEGFA ELISA; error bars represent s.e.m. for n = 2, otherwise n = 1.

(c) Differences in VEGFA expression observed with variable numbers of DmrA domains fused to either the amino-terminal or carboxy-terminal end of dCas9 (DmrA-dCas9 or dCas9-DmrA, respectively) and with the VP64 activation domain fused to the amino or carboxy-terminal end of DmrC (VP64-DmrC or DmrC-VP64, respectively). These experiments also included an expression vector expressing three sgRNAs targeted upstream of the VEGFA transcription start site. Gene activation assessed by VEGFA ELISA; error bars represent s.e.m. for n = 3.