Journal: ACS synthetic biology

Article Title: Rewiring Calcium Signaling for Precise Transcriptional Reprogramming

doi: 10.1021/acssynbio.7b00467

Figure Lengend Snippet: Design of genetically encoded CaRROT to enable spatiotemporal control of transcriptional reprogramming in mammals. This synthetic device is composed of (i) second-generation Opto-CRAC made of LOV2-SOAR chimeras that could photoactivate ORAI calcium channels on the plasma membrane with tight control over Ca2+ signals; and (ii) a calcium-responsive dCas9 fusion construct (e.g., NFAT1–460-dCas9-VP64). The N-terminal NFAT fragment used in the design lacks the C-terminal DNA binding domain to avoid binding to endogenous NFAT targets. In the dark, CaRROT stays in the cytosol. Upon blue light illumination, CaRROT undergoes light-inducible nuclear translocation due to the cleavage of the phosphate groups (P) by calcineurin to turn on gene expression at targeted loci in the presence of small guide RNAs (sgRNAs). In addition to light, chemicals or ligands that could elicit intracellular calcium mobilization could likewise rewire calcium signaling to achieve inducible transcriptional reprogramming at targeted genomic loci.

Article Snippet: EGFP reporter containing eight copies of a gRNA binding site for light-inducible dCas9 activation was obtained from Addgene (#60718).

Techniques: Construct, Binding Assay, Translocation Assay, Expressing

Journal: ACS synthetic biology

Article Title: Rewiring Calcium Signaling for Precise Transcriptional Reprogramming

doi: 10.1021/acssynbio.7b00467

Figure Lengend Snippet: Design and optimization of CaRROT and second-generation Opto-CRAC constructs to enable tight control of dCas9 nuclear translocation. (A) Design of dCas9-fUsion constructs for inducible nuclear translocation: (i) fusion with light-sensitive NLS signals (BiNLS: V1–V2); or (ii) through Ca2+-dependent nuclear translocation (V3–V5). (B) Opto-CRAC designed to photoinduce Ca2+ influx by optimizing STIM1-CT fragments, the linker and fusion to LOV2-binder Zdk. (C) Basal fluorescence intensities of GCaMP6s-HeLa cells transfected with indicated Opto-CRAC constructs in the dark. At least 30 cells were analyzed in the assay for each construct. (D) Light-inducible fold-change in the GCaMP6s fluorescence intensity (at 2 min postphotostimulation at 470 nm; 50 μW/cm2) in HeLa cells expressing the indicated second generation Opto-CRAC constructs. Data were shown as mean ± SD (n = 30 cells from three independent experiments). (E) Time course showing light-inducible increase of GCaMP6s signals in HeLa cells expressing Opto-CRAC-B10. Representative images showing GCaMP6s fluorescence before and after light stimulation were presented on the right. Data were showed as mean ± SD (n = 30 cells). (F) Monitoring light-inducible translocation of dCas9-VP64 or dCas9-NFAT1–460-VP64 from cytosol to nuclei in the same cells expressing the indicated constructs by confocal imaging. (G,H) Time course showing the fold-change of nuclear GFP intensity following blue light stimulation (G) and quantification of signals before and after light illumination for 30 min (H). Data were showed as mean ± SD (n = 9). Scale bar: 5 μm. ****P < 0.0001 compared to the dark group (two-tailed Student’s t-test).

Article Snippet: EGFP reporter containing eight copies of a gRNA binding site for light-inducible dCas9 activation was obtained from Addgene (#60718).

Techniques: Construct, Translocation Assay, Fluorescence, Transfection, Expressing, Imaging, Two Tailed Test

Journal: ACS synthetic biology

Article Title: Rewiring Calcium Signaling for Precise Transcriptional Reprogramming

doi: 10.1021/acssynbio.7b00467

Figure Lengend Snippet: CaRROT-mediated light-inducible activation of endogenous gene expression. Light-induced endogenous gene expression of (A) MYOD1 and (B) ASCL1 in HEK293T cells were measured by qRT-PCR. Cells were transfected with dCas9-NLS-VP64 as positive control (PC), BFP-tagged-CaRROT-V5 construct, Opto-CRAC-B10 and indicated sgRNAs or the empty plasmid (pTriEX-BFP). Cells were subjected to pulsed blue light stimulation (470 nm, 50 μW/cm2). *P < 0.05; ***P < 0.001; ****P < 0.0001 compared to the dark group (two-tailed Student’s t-test).

Article Snippet: EGFP reporter containing eight copies of a gRNA binding site for light-inducible dCas9 activation was obtained from Addgene (#60718).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Positive Control, Construct, Plasmid Preparation, Two Tailed Test