CRISPR Gene Editing in Stem Cells

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Epigenetic Regulation with CRISPR/dCas9
dCas9 fused to epigenetic-modifying enzymes has been used to introduce locus-specific
epigenetic modi fications in the genome. Examples include fusing dCas9 to the core catalytic
domain of the human acetyltransferase p300 (p300core
[2_TD$DIFF]), which allowed acetylation of histone H3
Lys27 (H3K27) and upregulation of genes when binding to proximal or distal enhancers [57];
fusing dCas9 to lysine demethylase 1 (LSD1) reduced the acetylation level of H3K27 [58]; fusing
dCas9 to KRAB increased the H3K9me3 mark near the target site [59]; and fusing dCas9 to the
DNA methyltransferase DNMT3A increased CpG methylation near the target site [60]. These
studies also demonstrated modified gene expression levels due to Cas9-mediated locus-
specific epigenetic modifications. For example, in mouse embryonic stem cells, the enhancers
of pluripotency factors, such as Oct4 and Tbx3, could be repressed by dCas9–LSD1 fusion,
leading to loss of pluripotency [58,61].
While these examples provide an approach to edit the epigenetic states of essentially any locus
in the genome, a largely unexplored question is the fate of the synthetic epigenetic marks, and
whether they can be stably inherited when cells proliferate. Furthermore, given the diverse types
of epigenetic modification and their mutual interactions, a comprehensive toolkit comprising
multiple orthogonally acting dCas9s and their cognate sgRNA that allows the flexible editing of
multiple epigenetic (histone or DNA) marks simultaneously is needed. Such a toolkit would be
useful for understanding the function of diverse epigenetic marks, their interactions, and their
relation to genomic and cellular functions.
Large-Scale Functional Genomic Studies Using CRISPR/Cas9
One of the powerful applications of the CRISPR/Cas9 technology is the high-throughput
screening of genomic functions. The oligo libraries encoding hundreds of thousands of sgRNAs
can be computationally designed and chemically synthesized to target a broad set of genome
sequences. By pairing with Cas9 or dCas9 fusion proteins, this provides an approach to
systematically knock out, repress, or activate genes on a large scale. The technique requires
a delicate delivery method that ensures that every cell only receives a single sgRNA, usually via
lentiviral or retroviral delivery into mammalian cells. The screens are frequently performed in a
pooled manner, because cells transduced with the lentiviral library as a mixed population are
cultured together. Via deep sequencing and analysis of the sgRNA features in the pooled cells,
genes causing changes in cell growth and death can be inferred with bioinformatics. Indeed,
CRISPR screens can easily identify genes, their regulatory elements, and protein domains in the
mammalian genome responsible for cell growth and drug resistance [62]. A genomic tiling
screen using CRISPR/Cas9 precisely mapped functional domains within enhancer elements
and found that a p53-bound enhancer of the p53 effector gene CDKN1A was required for
oncogene-induced senescence in immortalized human cells [63].
Using the endonuclease Cas9, loss-of-function genome-wide knockout screens have been
performed in cultured or primary mammalian cells with sgRNA libraries (usually three– ten
sgRNAs per gene) to investigate a range of phenotypes, including cell growth, cancer cell drug
resistance, and viral susceptibility [64–66]. A genome-scale sgRNA library can also be used to
manipulate cultured cells that are later introduced in vivo. Indeed, a genome-scale sgRNA library
was created to mutagenize a non-metastatic mouse cancer cell line for the study of metastasis in
a mouse model [67]. The mutant cell pool rapidly generated metastases when transplanted into
immunocompromised mice in vivo. Sequencing of the metastatic cells suggested genes that
accelerate lung cancer metastases and development of late-stage primary tumors. Moreover,
this screening method can be extended to use in primary cells, which can lead to novel findings
that are often overlooked using cell lines. Indeed, introducing a genome-wide sgRNA library into
primary dendritic cells (DCs) allowed for the identification of genes related to cell growth that
induce tumor necrosis factor (TNF) in response to bacterial lipopolysaccharide (LPS), an
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