RNA-guided gene activation by CRISPR-Cas9–based transcription factors


Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

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Figure 1: RNA-guided activation of the human IL1RN gene by dCas9-VP64.
Figure 2: RNA-guided activation of human genes.

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This work was supported by a US National Institutes of Health (NIH) Director's New Innovator Award (DP2OD008586), National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award (CBET-1151035), NIH R03AR061042, and an American Heart Association Scientist Development Grant (10SDG3060033) to C.A.G., and grants from the NIH to G.E.C. (U54HG004563), K.W.L. (EB015300 and HL109442), and F.G. (R01AR48852). K.A.G. and P.I.T. were supported by NSF Graduate Research Fellowships. L.R.P. was supported by an NIH Biotechnology Training Grant to the Duke Center for Biomolecular and Tissue Engineering (T32GM008555). D.G.O. was supported by a predoctoral fellowship from the American Heart Association. C. Grigsby (Duke University) provided the pABOL polymer used in MEF transfections.

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P.P., C.M.V., A.F.A., D.G.O., G.E.C., T.E.R. and C.A.G. designed experiments. P.P., D.D.K., C.M.V., A.F.A., A.M.K., L.R.P., P.I.T., K.A.G. and D.G.O. performed the experiments. P.P., D.D.K., C.M.V., A.F.A., A.M.K., L.R.P., P.I.T., K.A.G., D.G.O., K.W.L., F.G., G.E.C., T.E.R. and C.A.G. analyzed the data. P.P. and C.A.G. wrote the manuscript.

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Correspondence to Charles A Gersbach.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, and Supplementary Tables 1 and 2 (PDF 1133 kb)

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Perez-Pinera, P., Kocak, D., Vockley, C. et al. RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nat Methods 10, 973–976 (2013). https://doi.org/10.1038/nmeth.2600

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