Highly efficient Cas9-mediated transcriptional programming (2024)

Fluorescent reporter assay quantifying the amount of transcriptional activation for the various dCas9 fusion proteins. The particular activation domain, mediator complex member or RNA polymerase subunit fused to the C terminus of dCas9 is listed. The tested activation domains represent minimal activation domains. Mediator and RNA polymerase members fused to dCas9 were full length cDNAs. Data are shown as mean fluorescence ± s.e.m., n = 2 independent transfections.

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(a) Transcriptional activation via Cas9 was performed by fusing activation domains to the C terminus of a nuclease-null dCas9 protein. The tripartite VPR activator consisting of VP64-p65-Rta activation domains fused in tandem to dCas9, is illustrated. (b) Fluorescent reporter assay quantifying the amount of activation from the various dCas9 domain assemblies. Data are shown as median fluorescence ± s.e.m. n = 5 independent transfections. * denotes significance of dCas9-VP64-p65-Rta over all constructs including Reporter Control, P = <0.0001.

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Fluorescent reporter assay quantifying the effects of substituting each member of the VPR complex with the mCherry fluorescent protein compared to VP64 and the intact VPR complex. Data are shown as median fluorescence ± s.e.m. n = 5 independent transfections. * denotes significance of dCas9-VP64-p65-RTA over all constructs including Reporter Control, P = <0.0001.

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Fluorescent reporter assay quantifying the activation potential of each of the different non-repeating combinations between VP64, p65 and Rta. The activation domain fused to dCas9 is listed. Data are shown as median fluorescence ± s.e.m. n = 5 independent transfections. * denotes significance of dCas9-VP64-p65-Rta over all constructs including Reporter Control, P = <0.005.

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(a) Fluorescent reporter assay quantifying the level of transcriptional activation from Streptococcus thermophilus, ST1-dCas9-VP64 and ST1-dCas9-VPR proteins. Data are shown as median fluorescence ± s.e.m. n = 3 independent transfections. For *, P = <0.001. Difference between ST1-dCas9-VP64 vs. ST-dCas9-VPR is significant, P = <0.0001 (b) Fluorescent reporter assay quantifying transcriptional activation for designer transcription activator like effector, TALE-VP64 and TALE-VPR proteins. Data are shown as median fluorescence ± s.e.m. n = 3 independent transfections. For *, P = <0.005. Difference between TALE-VP64 vs. TALE-VPR is significant, P = <0.0005 (c) Fluorescent reporter assay quantifying the level of transcriptional activation for zinc-finger protein (ZNF) fused to either VP64 or VPR. Data are shown as median fluorescence ± s.e.m. n = 3 independent transfections. For *, P = <0.01. Difference between ZNF-VP64 vs. ZNF-VPR is significant, P = <0.05.

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Fold activation (y-axis) is calculated by measuring the level of target expression above background, when indicated gene is activated with dCas9-VPR. Basal expression level (x-axis) is calculated by measuring basal target gene expression relative to ß-actin. For all data points n = 3 independent transfections.

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(a) Levels of RNA expression for the neuronal targets NEUROD1, NEUROG2, and ASCL1 in non-activated HEK 293Ts, VPR activated HEK 293Ts, and human brain tissue – target expression is calculated relative to ß-actin level within each sample (b) Levels of RNA expression for the cardiac targets TTN, ACTC1, and MIAT in non-activated HEK 293Ts, VPR activated HEK 293Ts, and human heart tissue – all relative to ß-actin level within each sample. (c) Relative levels of RHOXF2 transcript expressed in non-activated HEK 293Ts, VPR activated HEK 293Ts, and human testes tissue – all relative to ß-actin level within each sample. For all 293T data, n = 3 independent transfections, for human tissue samples n = 1 total RNA extract.

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(a) RNA expression of individual targets within S. cerevisiae containing the indicated activator along with a gRNA against either GAL7, HED1 or a control guide with no genomic target. Data are shown as the mean ± s.e.m (n = 3 independent colonies for GAL7 and n = 4 independent colonies for HED1). For *, P = <0.01. (b) RNA expression of individual targets in D. melanogaster S2R+ cells, transfected with the indicated dCas9 activator and guide RNAs against the fly genes Metchnikowin (Mtk) or Cecropin-A1 (CecA1). Data are shown as the mean ± s.e.m (n = 3 independent transfections). For *, P = <0.05. (c) RNA expression of individual targets within M. musculus Neuro-2A cells transfected with the indicated dCas9 activator along with gRNAs targeting either Acta1, Actc1, Ttn, or Tuna. Data are shown as the mean ± s.e.m (n = 3 independent transfections). For *, P = <0.01 (n.s. = not significant). Comparisons between VP64 and VPR within all panels are significant, P = <0.05.

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(a) Pseudocolored immunofluorescence images for NucBlue (blue, total cells) and neurofilament 200 (red, iNeurons). Images were taken 4 days after doxycycline induction and are representative of biological triplicates (separately seeded wells). Scale bar represents 100 µm. (b) Immunofluorescence quantification and comparison of iNeurons generated by either dCas9-VP64 or dCas9-VPR. Data are shown as the mean ± s.e.m. (n = 3 independent platings of stable cell lines, with each replicate being an average of 24 separate images). For *, P = < 0.001.

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