CRISPR Gene Editing in Stem Cells

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Cell Stem Cell
Short Article
Targeted Epigenetic Remodeling of Endogenous Loci
by CRISPR/Cas9-Based Transcriptional Activators
Directly Converts Fibroblasts to Neuronal Cells
Joshua B. Black,1 Andrew F. Adler,1,9 Hong-Gang Wang,6,8,10 Anthony M. D’Ippolito,2,3 Hunter A. Hutchinson,1
Timothy E. Reddy,2,4 Geoffrey S. Pitt,6,7,8 Kam W. Leong,1,11 and Charles A. Gersbach1,2,5, *
1Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
2Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA
3University Program in Genetics and Genomics, Duke University Medical Center, Durham, NC 27710, USA
4Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA
5Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USA
6Ion Channel Research Unit, Duke University Medical Center, Durham, NC 27710, USA
7Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA
8Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
9Present address: Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA
10 Present address: Cardiovascular Research Institute, Weill Cornell Medicine, New York, NY 10021, USA
11 Present address: Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA
*Correspondence:
SUMMARY
Overexpression of exogenous fate-specifying tran-
scription factors can directly reprogram differ-
entiated somatic cells to target cell types. Here,
we show that similar reprogramming can also be
achieved through the direct activation of endoge-
nous genes using engineered CRISPR/Cas9-based
transcriptional activators. We use this approach to
induce activation of the endogenous Brn2, Ascl1,
and Myt1l genes (BAM factors) to convert mouse em-
bryonic fibroblasts to induced neuronal cells. This
direct activation of endogenous genes rapidly re-
modeled the epigenetic state of the target loci and
induced sustained endogenous gene expression
during reprogramming. Thus, transcriptional activa-
tion and epigenetic remodeling of endogenous mas-
ter transcription factors are sufficient for conversion
between cell types. The rapid and sustained activa-
tion of endogenous genes in their native chromatin
context by this approach may facilitate reprogram-
ming with transient methods that avoid genomic inte-
gration and provides a new strategy for overcoming
epigenetic barriers to cell fate specification.
INTRODUCTION
Direct reprogramming of somatic cells has tremendous potential
to advance applications in disease modeling, drug discovery,
and gene and cell therapies. Common approaches to achieve
cellular reprogramming rely on the ectopic expression of trans-
genes encoding lineage-specific transcription factors (Davis
et al., 1987; Takahashi and Yamanaka, 2006; Vierbuchen
et al., 2010). To demonstrate stable cellular reprogramming to
an autonomous cell phenotype, the expression of exogenous
transcription factors should be transient. Thus the establishment
of positive feedback networks regulating endogenous genes
is necessary to sustain a transgene-independent cellular iden-
tity (Vierbuchen and Wernig, 2011). In many cases, the endoge-
nous genes are occluded by cis-acting repressive chromatin
marks that are slow to remodel (Vierbuchen and Wernig, 2012).
This slow remodeling process typically necessitates prolonged
expression of the exogenous factors, limiting the efficacy of
transient delivery methods, and poses a major bottleneck to
improving the efficiency, speed, and robustness of reprogram-
ming (Hanna et al., 2009).
The type II clustered regularly interspaced short palindromic
repeat (CRISPR) system and the CRISPR-associated Cas9
nuclease have recently been repurposed from an adaptive im-
mune system in bacteria and archaea to a gene editing tool
(Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013b) and tran-
scriptional regulator (Cheng et al., 2013; Gilbert et al., 2013; Ko-
nermann et al., 2013; Maeder et al., 2013b; Mali et al., 2013a;
Perez-Pinera et al., 2013; Qi et al., 2013) of endogenous genes
in mammalian cells. The ability to program these transcription
factors to target any genomic locus of interest through the simple
exchange of the 20-nt targeting sequence of the guide RNA
(gRNA) enables a simple, robust, and highly scalable method
for control of complex transcriptional networks (Thakore et al.,
2016). Furthermore, dCas9-based transcription factors can
target stably silenced genes within compacted chromatin to
initiate chromatin remodeling and transcriptional activation
(Perez-Pinera et al., 2013; Polstein et al., 2015). Thus, this tech-
nology may provide a method to deterministically initiate expres-
sion of endogenous gene networks of alternate cell lineages.
The CRISPR/Cas9 system and other platforms for program-
mable transcriptional regulation have been incorporated into
methods for cellular reprogramming in a few recent studies.
Gao et al. used transcription activator-like effector (TALE)-based
406 Cell Stem Cell 19 , 406–414, September 1, 2016 ª 2016 Elsevier Inc.