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expression or activity. This type of tool could potentially be used to create a perma-
nent record of cell signaling inputs occurring throughout development.
A proof-of-principle study published recently established the possibility of aCas9-based recording device [ 156 ]. Using an stgRNA approach coupled with an
NFκB-responsive element to link Cas9 expression with NFκB activity, Cas9-
induced mutation of the stgRNA cassette was detected in response to inflammation,
demonstrating that a transient signal can be permanently recorded in the DNA. On
a population level, induction of inflammation by varying amounts of stimulus
resulted in mutation of the stgRNA cassette such that increased strength and/or
duration of signal resulted in increased mutation; however, because of the difficulty
in precisely controlling and/or predicting the mutation event in response to Cas9
cleavage, it is not yet feasible to directly translate mutational load to signal intensity
and/or duration on a single-cell level. This would require first creating a calibration
metric by generating a transition probability matrix for each gRNA—a process that
could potentially vary depending on cell-type and cell-cycle state and the favored
repair mechanisms associated with each. In addition, as was seen in Kalhor et al.,
the use of stgRNAs necessitates the use of long gRNAs to compensate for the pro-
pensity of Cas9 DSBs to result in deletions [ 155 ].
3.5.2 CRISPR GE and Ex Vivo Organogenesis
The prior perspectives have emphasized the importance of the output of whole net-
works in regulating cell-identity during development. However, they largely main-
tained their focus on mechanisms occurring within a single cell, whereas the
development of whole tissues and organs involves the co-development of distinct
cell types not as autonomous units but rather as parts of a whole with complex inter-
relationships. A complete view of development, thus, relies on an understanding of
how the external environment, including the intercellular network, guides develop-
ment, with particular emphasis on how it is coupled with cell-internal genome and
epigenome regulatory networks to maintain cell- and tissue-identity.
The use of directed differentiation experiments in vitro, which use growth and/or signaling factors in the culture medium to guide the development of particular
lineages from pluripotent stem cells (PSCs, either embryonic (ESCs), or repro-
grammed (iPSCs, [ 160 ])), are useful tools to ask developmental questions at the
level of a single cell, but are poor representations of the intercellular communica-
tion involved in tissue development. Recent developments in 3D–culture sys-
tems—using 3D matrices as a surrogate extracellular matrix (ECM)—push beyond
traditional 2D cultures to better mimic the diversity of cell types and interactions
within a developing tissue environment (reviewed in [ 161 – 166 ]). Termed ‘organ-
oids,’ these 3D mini-organs resemble their in vivo counterpart in composition,
structure and (at least some) function. They can be derived from PSCs (as well as
neonatal tissue stem cells and adult stem cells (AdSCs)), which after initial stimu-
lation toward the desired germ layer and subsequent lineage, largely form through
R.K. Delker and R.S. Mann