CRISPR Gene Editing in Stem Cells

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delivered Cas9 may only edit genes in a fraction of cells, and the approach may be most effective
for studying the effects of loss-of-function mutations on cell autonomous properties.
Genome Imaging Using CRISPR/Cas9
Imaging offers a direct approach for studying the spatial and temporal behavior of the genome in
living cells [92]. The ability of Cas9 to target specific sequences in the genome makes it a
promising imaging tool for directly observing genomic organization and dynamics in cells. The
first proof-of-concept work fused the S. pyogenes dCas9 to EGFP and used the fusion protein
to visualize the dynamics of coding or noncoding sequences in living human cell lines [93]. The
authors tracked the dynamics of telomeres, and the repetitive and nonrepetitive sequences of
coding genes ( MUC4 and MUC1) in a short time frame ( minutes) and throughout the whole cell
cycle. In addition, dCas9 fused to EGFP has been used to label endogenous centromeres and
telomeres loci in live mouse embryonic stem cells [94]. The development of the SunTag system,
a repeating peptide array that can recruit multiple copies of an antibody-fusion protein,
enhanced the sensitivity to amplify the dCas9 fluorescent signal in the genome [53]. Using
dCas9 orthologs tagged with different fluorescent proteins, it was shown that the dynamics of
multiple repetitive genomic loci could be tracked in living cells [95]. A method termed ‘Cas9-
mediated fluorescence in situ hybridization’ (CASFISH) further combined dCas9 with fluores-
cencein situ hybridization (FISH) [96]. Due to the specific DNA targeting and unwinding activity of
dCas9, CASFISH is a fast and convenient process for labeling DNA elements while avoiding
treatment of heat and disruptive chemicals that distort the natural organization of the nucleus,
which is normally seen in FISH. Thus, the approach preserves the spatial relations of the genetic
elements that are important for studying gene expression.
Recent work also established a CRISPR approach to facilitate super-resolution imaging in living
mammalian cells [97]. Current live cell super-resolution imaging normally relies on the overexpres-
sion of a host protein fused to a fluorescent protein, which results in artifacts that may obscure the
interpretation of imaging results. Using CRISPR/Cas9 to fluorescently tag the endogenous genes
that are expressed from their native genomic loci could allow genes to be expressed at close to
endogenous levels, thus avoiding artifacts. Based on this idea, a method termed ‘reversible
saturable optical fluorescence transitions’ (RESOLFT) was developed, wherein heterozygous
and homozygous Cas9-edited human knock-in cell lines were generated that expressed the
reversibly switchable fluorescent protein rsEGFP2 from their respective native genomes, which
prevented the appearance of typical overexpression-induced artifacts in these cells.
To enhance signals for endogenous proteins imaging, one study adapted self-complementing
split fl uorescent proteins, GFP11 and sfCherry11, derived from the sfGFP and sfCherry [98]. The
small sizes of these split fluorescent domains (16–18 amino acids) enable them to be easily
inserted into endogenous genomic loci via CRISPR gene editing. Tandem arrays of these
domains further amplify fluorescence signals in imaging, such as for tracking intraflagellar
transport particles.
In addition to DNA imaging, S. pyogenes dCas9 can also allow for endogenous RNA imaging in
living cells [99]. In the presence of sgRNAs targeting mRNA and a stabilized PAMmer oligonu-
cleotide that contains the PAM domain for dCas9 binding, specifically targeted RNA can be
visualized. Indeed, it was observed that nuclear localized dCas9 could be exported to the
cytoplasm. Furthermore, dCas9 allowed for tracking of RNA during induced RNA/protein
accumulation in the presence of oxidative stress.
Lineage Tracing Using CRISPR/Cas9
Gene editing has been used as tools for cell lineage tracing. One recent study demonstrated a
lineage-tracing method termed ‘ genome editing of synthetic target arrays for lineage tracing’
884 Trends in Cell Biology, November 2016, Vol. 26, No. 11