Fig. 2
From: Engineering cell signaling using tunable CRISPR–Cpf1-based transcription factors
The dAsCpf1-based transcriptional activators effectively increased transcription. a Design of the dAsCpf1-based transcriptional activator. To generate a fusion protein capable of transcriptional activation, we directly tethered the tripartite VP64 to the C terminus of dAsCpf1. To generate a fusion protein capable of mediating stronger transcriptional activation, we also tethered the tripartite VPR activator to the C terminus of dAsCpf1. VPR is a fusion of VP64, p65, and RTA. The fusion protein was bound to the crRNA and formed a transcriptional activation complex. b Locations of crRNAs targeted to the gene promoter. Blue lines indicate base pairing nucleotides of the crRNAs that bound to either the template DNA strand or the non-template DNA strand. c The dAsCpf1-based transcriptional activators displayed RNA-guided transcriptional activation as detected by fluorescent microscopy. Representative images of the transfected cells are shown. Scale bar, 1000 μm. d For DNMT1, we designed three independent crRNAs and crRNA arrays expressing all the possible pairs of these crRNAs, and assayed transcriptional activation by qRT-PCR. Reported data are the mean ± SD from five independent experiments. ** P < 0.01 compared to the non-target crRNA control, by paired, one-sided t-tests
