70 128
transfections and providing the pUNO-Brn2 plasmid; Rui Dai for technical
assistance; and Jeff Stogsdill and Cagla Eroglu for supplying astrocytes and
providing protocols for co-culture experiments. This work was supported by
US NIH grants to C.A.G. and T.E.R. (R01DA036865 and U01HG007900),
an NIH Director’s New Innovator Award (DP2OD008586) and National Sci-
ence Foundation (NSF) Faculty Early Career Development (CAREER) Award
(CBET-1151035) to C.A.G., and NIH grant P30AR066527. J.B.B. was sup-
ported by an NIH Biotechnology Training Grant (T32GM008555).
Received: May 13, 2015
Revised: May 11, 2016
Accepted: June 30, 2016
Published: August 11, 2016
REFERENCES
Adler, A.F., Grigsby, C.L., Kulangara, K., Wang, H., Yasuda, R., and Leong,
K.W. (2012). Nonviral direct conversion of primary mouse embryonic fibro-
blasts to neuronal cells. Mol. Ther. Nucleic Acids
1
, e32.
Balboa, D., Weltner, J., Eurola, S., Trokovic, R., Wartiovaara, K., and
Otonkoski, T. (2015). Conditionally stabilized dCas9 activator for controlling
gene expression in human cell reprogramming and differentiation. Stem Cell
Reports
5
, 448–459.
Chakraborty, S., Ji, H., Kabadi, A.M., Gersbach, C.A., Christoforou, N., and
Leong, K.W. (2014). A CRISPR/Cas9-based system for reprogramming cell
lineage specification. Stem Cell Reports
3
, 940–947.
Chavez, A., Scheiman, J., Vora, S., Pruitt, B.W., Tuttle, M., P R Iyer, E., Lin, S.,
Kiani, S., Guzman, C.D., Wiegand, D.J., et al. (2015). Highly efficient Cas9-
mediated transcriptional programming. Nat. Methods
12
, 326–328.
Cheng, A.W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T.W.,
Rangarajan, S., Shivalila, C.S., Dadon, D.B., and Jaenisch, R. (2013).
Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided
transcriptional activator system. Cell Res.
23
, 1163–1171.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X.,
Jiang, W., Marraffini, L.A., and Zhang, F. (2013). Multiplex genome engineering
using CRISPR/Cas systems. Science
339
, 819–823.
Mouse ENCODE Consortium (2012). An encyclopedia of mouse DNA elements
(Mouse ENCODE). Genome Biol.
13
, 418.
Davis, R.L., Weintraub, H., and Lassar, A.B. (1987). Expression of a single
transfected cDNA converts fibroblasts to myoblasts. Cell
51
, 987–1000.
Gao, X., Yang, J., Tsang, J.C., Ooi, J., Wu, D., and Liu, P. (2013).
Reprogramming to pluripotency using designer TALE transcription factors
targeting enhancers. Stem Cell Reports
1
, 183–197.
Gao, X., Tsang, J.C., Gaba, F., Wu, D., Lu, L., and Liu, P. (2014). Comparison
of TALE designer transcription factors and the CRISPR/dCas9 in regulation
of gene expression by targeting enhancers. Nucleic Acids Res.
42
, e155.
Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-
Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., et al. (2013).
CRISPR-mediated modular RNA-guided regulation of transcription in eukary-
otes. Cell
154
, 442–451.
Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner, C.J., Creyghton, M.P.,
van Oudenaarden, A., and Jaenisch, R. (2009). Direct cell reprogramming is
a stochastic process amenable to acceleration. Nature
462
, 595–601.
Hilton, I.B., D’Ippolito, A.M., Vockley, C.M., Thakore, P.I., Crawford, G.E.,
Reddy, T.E., and Gersbach, C.A. (2015). Epigenome editing by a CRISPR-
Cas9-based acetyltransferase activates genes from promoters and en-
hancers. Nat. Biotechnol.
33
, 510–517.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier,
E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity. Science
337
, 816–821.
Kabadi, A.M., Ousterout, D.G., Hilton, I.B., and Gersbach, C.A. (2014).
Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral
vector. Nucleic Acids Res.
42
, e147.
Kearns, N.A., Pham, H., Tabak, B., Genga, R.M., Silverstein, N.J., Garber, M.,
and Maehr, R. (2015). Functional annotation of native enhancers with a Cas9-
histone demethylase fusion. Nat. Methods.
12
, 401–413.
Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., Kim, J., Aryee, M.J., Ji,
H., Ehrlich, L.I., et al. (2010). Epigenetic memory in induced pluripotent stem
cells. Nature
467
, 285–290.
Konermann, S., Brigham, M.D., Trevino, A.E., Hsu, P.D., Heidenreich, M.,
Cong, L., Platt, R.J., Scott, D.A., Church, G.M., and Zhang, F. (2013).
Optical control of mammalian endogenous transcription and epigenetic states.
Nature
500
, 472–476.
Ladewig, J., Mertens, J., Kesavan, J., Doerr, J., Poppe, D., Glaue, F., Herms, S.,
Wernet, P., Ko¨ gler, G., Mu¨ ller, F.J., et al. (2012). Small molecules enable highly
efficient neuronal conversion of human fibroblasts. Nat. Methods
9
, 575–578.
Maeder, M.L., Angstman, J.F., Richardson, M.E., Linder, S.J., Cascio, V.M.,
Tsai, S.Q., Ho, Q.H., Sander, J.D., Reyon, D., Bernstein, B.E., et al. (2013a).
Targeted DNA demethylation and activation of endogenous genes using pro-
grammable TALE-TET1 fusion proteins. Nat. Biotechnol.
31
, 1137–1142.
Maeder, M.L., Linder, S.J., Cascio, V.M., Fu, Y., Ho, Q.H., and Joung, J.K.
(2013b). CRISPR RNA-guided activation of endogenous human genes. Nat.
Methods
10
, 977–979.
Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S.,
Yang, L., and Church, G.M. (2013a). CAS9 transcriptional activators for target
specificity screening and paired nickases for cooperative genome engineer-
ing. Nat. Biotechnol.
31
, 833–838.
Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E.,
and Church, G.M. (2013b). RNA-guided human genome engineering via Cas9.
Science
339
, 823–826.
Mendenhall, E.M., Williamson, K.E., Reyon, D., Zou, J.Y., Ram, O., Joung, J.K.,
and Bernstein, B.E. (2013). Locus-specific editing of histone modifications at
endogenous enhancers. Nat. Biotechnol.
31
, 1133–1136.
Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Adler, A.F., Kabadi, A.M.,
Polstein, L.R., Thakore, P.I., Glass, K.A., Ousterout, D.G., Leong, K.W., et al.
(2013). RNA-guided gene activation by CRISPR-Cas9-based transcription
factors. Nat. Methods
10
, 973–976.
Polstein, L.R., Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Bledsoe, P.,
Song, L., Safi, A., Crawford, G.E., Reddy, T.E., and Gersbach, C.A. (2015).
Genome-wide specificity of DNA binding, gene regulation, and chromatin re-
modeling by TALE- and CRISPR/Cas9-based transcriptional activators.
Genome Res.
25
, 1158–1169.
Qi, L.S., Larson, M.H., Gilbert, L.A., Doudna, J.A., Weissman, J.S., Arkin, A.P.,
and Lim, W.A. (2013). Repurposing CRISPR as an RNA-guided platform for
sequence-specific control of gene expression. Cell
152
, 1173–1183.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells
from mouse embryonic and adult fibroblast cultures by defined factors. Cell
126
, 663–676.
Thakore, P.I., Black, J.B., Hilton, I.B., and Gersbach, C.A. (2016). Editing the
epigenome: technologies for programmable transcription and epigenetic
modulation. Nat. Methods
13
, 127–137.
Treutlein, B., Lee, Q.Y., Camp, J.G., Mall, M., Koh, W., Shariati, S.A., Sim, S.,
Neff, N.F., Skotheim, J.M., Wernig, M., and Quake, S.R. (2016). Dissecting
direct reprogramming from fibroblast to neuron using single-cell RNA-seq.
Nature
534
, 391–395.
Vierbuchen, T., and Wernig, M. (2011). Direct lineage conversions: unnatural
but useful? Nat. Biotechnol.
29
, 892–907.
Vierbuchen, T., and Wernig, M. (2012). Molecular roadblocks for cellular re-
programming. Mol. Cell
47
, 827–838.
Vierbuchen, T., Ostermeier, A., Pang, Z.P., Kokubu, Y., Su¨ dhof, T.C., and
Wernig, M. (2010). Direct conversion of fibroblasts to functional neurons by
defined factors. Nature
463
, 1035–1041.
Wei, S., Zou, Q., Lai, S., Zhang, Q., Li, L., Yan, Q., Zhou, X., Zhong, H., and Lai,
L. (2016). Conversion of embryonic stem cells into extraembryonic lineages by
CRISPR-mediated activators. Sci. Rep.
6
, 19648.
414
Cell Stem Cell
19
, 406–414, September 1, 2016