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Highly efficient Cas9-mediated transcriptional programming

Abstract

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

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Figure 1: Gene activation using VPR.
Figure 2: dCas9-mediated iPSC neuronal differentiation using VPR.

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Acknowledgements

We thank K. Esvelt, P. Mali and the rest of the members of the Church and Collins labs for helpful discussions. We thank T. Ferrante, S. Byrne and M. Farrell for technical assistance, A. Keung (Massachusetts Institute of Technology) for providing zinc-finger reporter constructs, and J. Schulak for graphic design. This work was supported by US National Institutes of Health National Human Genome Research Institute grant P50 HG005550, US Department of Energy grant DE-FG02-02ER63445 and the Wyss Institute for Biologically Inspired Engineering. A.C. acknowledges funding by the National Cancer Institute grant 5T32CA009216-34. S.V. acknowledges funding by the National Science Foundation Graduate Research Fellowship Program, the Department of Biological Engineering at the Massachusetts Institute of Technology and the Department of Genetics at Harvard Medical School.

Author information

Authors and Affiliations

Authors

Contributions

A.C. and J.S. conceived of the study. A.C., J.S. and S.V. designed and performed experiments and interpreted data. B.W.P. designed and developed fusion libraries. M.T., D.T.-O., C.D.G. and D.J.W. performed experiments. N.D. and J.L.B. developed reagents. E.P.R.I. performed the iNeuron image analysis. S.K. and R.W. designed, tested and analyzed the TALE activator data. S.L., B.E.H. and N.P. designed, tested and analyzed the S2R+ cell experiments. J.A. designed a subset of the gRNAs. J.J.C. and G.M.C. supervised the study. A.C., J.S., S.V. and B.W.P. wrote the manuscript with support from M.T. and all other authors.

Corresponding author

Correspondence to George M Church.

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Competing interests

G.M.C. is a founding member of Editas Medicine, a company that applies genome editing technologies.

Integrated supplementary information

Supplementary Figure 1 dCas9 transcriptional reporter design.

(a) Fluorescent reporter construct consists of a tdTomato or sfGFP reporter downstream of a minimal CMV promoter, with an upstream dCas9 binding site (sequence of protospacer and PAM (GGG) are shown, separated by a space), tdTomato version of the reporter is illustrated. (b) Fluorescence microscopy images of HEK 293T cells all transfected with dCas9 reporter and the corresponding guide RNA along with the indicated dCas9 activator. Scale bar represents 100 µm.

Supplementary Figure 2 Targeted screen to identify activation domains that function with dCas9.

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.

Source data

Supplementary Figure 3 Serial fusion of activation domains to dCas9.

(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.

Source data

Supplementary Figure 4 Determining the essentiality of all VPR components.

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.

Source data

Supplementary Figure 5 Testing the effects of VP64, p65 and Rta order on activation.

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.

Source data

Supplementary Figure 6 Testing VPR activity when fused to other programmable DNA binding proteins.

(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.

Source data

Supplementary Figure 7 Efficiency of VPR mediated activation as a function of basal expression.

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.

Source data

Supplementary Figure 8 Comparison of VPR activated gene expression to that of native tissue.

(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.

Source data

Supplementary Figure 9 dCas9-VPR activity within various model organisms.

(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.

Source data

Supplementary Figure 10 Generation of iNeurons by NGN2 activation.

Bright field images of dCas9-AD NGN2 mediated iNeurons, four days after doxycycline addition. Red arrows point towards the cell body of iNeurons. Scale bar represents 100 µm.

Supplementary Figure 11 Generation of iNeurons by NEUROD1 activation.

Bright field images of dCas9-AD NEUROD1 mediated iNeurons, four days after doxycycline addition. Yellow arrows point towards the cell body of partially differentiated iPSCs. Red arrows point towards the cell body of iNeurons. Scale bar represents 100 µm.

Supplementary Figure 12 dCas9 mediated differentiation as determined by neurofilament 200 (NF200) staining.

(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.

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Table 1 and Supplementary Note (PDF 572 kb)

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Chavez, A., Scheiman, J., Vora, S. et al. Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12, 326–328 (2015). https://doi.org/10.1038/nmeth.3312

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