Highly efficient Cas9-mediated transcriptional programming

doi:10.1038/nmeth.3312

. 2015 Apr;12(4):326-8.

doi: 10.1038/nmeth.3312. Epub 2015 Mar 2.

Highly efficient Cas9-mediated transcriptional programming

Alejandro Chavez¹, Jonathan Scheiman², Suhani Vora³, Benjamin W Pruitt⁴, Marcelle Tuttle⁴, Eswar P R Iyer², Shuailiang Lin⁵, Samira Kiani⁶, Christopher D Guzman⁴, Daniel J Wiegand⁴, Dmitry Ter-Ovanesyan², Jonathan L Braff⁴, Noah Davidsohn², Benjamin E Housden⁷, Norbert Perrimon⁷, Ron Weiss⁸, John Aach⁹, James J Collins¹⁰, George M Church²

Affiliations

¹ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [3] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
² 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
³ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁴ Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
⁵ 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA. [3] School of Life Sciences, Tsinghua University, Beijing, China.
⁶ 1] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁷ 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.
⁸ 1] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁹ Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
¹⁰ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Institute for Medical Engineering &Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [4] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

PMID: 25730490
PMCID: PMC4393883
DOI: 10.1038/nmeth.3312

Free PMC article

Highly efficient Cas9-mediated transcriptional programming

Alejandro Chavez et al. Nat Methods. 2015 Apr.

Free PMC article

. 2015 Apr;12(4):326-8.

doi: 10.1038/nmeth.3312. Epub 2015 Mar 2.

Authors

Affiliations

¹ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. [3] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
² 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
³ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁴ Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
⁵ 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA. [3] School of Life Sciences, Tsinghua University, Beijing, China.
⁶ 1] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁷ 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.
⁸ 1] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
⁹ Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
¹⁰ 1] Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA. [2] Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Institute for Medical Engineering &Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [4] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

PMID: 25730490
PMCID: PMC4393883
DOI: 10.1038/nmeth.3312

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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).

Conflict of interest statement

Competing Financial Interests:

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

Figures

Figure 1

Gene activation using VPR. (a) RNA expression of individual targets in HEK 293T cells transfected simultaneously with three or four gRNAs targeting the indicated genes along with the labeled dCas9-activator construct. Negative controls (Neg.) were transfected with indicated guide RNAs alone. Data are shown as the mean ± s.e.m (n = 3 independent transfections). For *, P = < 0.05 (n.s. = not significant). Comparison of dCas9-VP64 vs. dCas9-VPR, for all genes, is significant (P = <0.0011). (b) RNA expression during multiplex activation of the indicated four endogenous gene targets. Data are shown as the mean ± s.e.m (n = 3 independent transfections). For *, P = < 0.05. Comparison of dCas9-VP64 vs. dCas9-VPR, for all genes, is significant (P = <0.0022).

Figure 2

dCas9-mediated iPSC neuronal differentiation using VPR. (a) Pseudocolored immunofluorescence images for NucBlue (blue, total cells) and beta III tubulin (red, iNeurons) four days after doxycycline induction. Images 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 is 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. (c) qRT-PCR analysis of mRNA expression levels of NGN2 and NEUROD1 in dCas9-AD iPS cell lines. Data is normalized to dCas9-VP64 cells and shown as the mean ± s.e.m. (n = 2 independent platings of each stable cell line). For *, P = < 0.05.

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References

1. Garneau JE, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67–71. - PubMed
1. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. - PMC - PubMed
1. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol Read Engl. 2009;155:733–740. - PubMed
1. Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. - PMC - PubMed
1. Mali P, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. - PMC - PubMed

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[1] Garneau JE, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67–71. - PubMed

[2] Garneau JE, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67–71. - PubMed

[3] Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. - PMC - PubMed

[4] Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. - PMC - PubMed

[5] Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol Read Engl. 2009;155:733–740. - PubMed

[6] Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol Read Engl. 2009;155:733–740. - PubMed

[7] Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. - PMC - PubMed

[8] Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. - PMC - PubMed

[9] Mali P, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. - PMC - PubMed

[10] Mali P, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. - PMC - PubMed

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Highly efficient Cas9-mediated transcriptional programming

Affiliations

Highly efficient Cas9-mediated transcriptional programming

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Abstract

Conflict of interest statement

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Cited by 313 articles

References

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Molecular Biology Databases

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Abstract

Conflict of interest statement

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Cited by 313 articles

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