CRISPR Gene Editing in Stem Cells

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CRISPR/Cas9 HDR, we generated an 11 nt deletion in AGIL (de-
noted bvhtdAGIL) at the endogenous Bvht locus in mESCs to
disrupt this loop (Figures 2A and 2B). We used a dual selection
strategy to facilitate recovery of homozygous clones ( 20%–
50% frequency) and expanded several clones for experimental
evaluation.
SHAPE probing of bvhtdAGIL RNA shows deletion of the AGIL
motif does not destabilize overall Bvht structure (Figures 2C
and S2A). Thus, we next examined Bvht levels in ESCs and found
that the mutant transcript was expressed at comparable levels to
wild-type (WT) by northern blot and qRT-PCR (Figures 2D and
2E). Similar to shRNA-mediated depletion of Bvht (Klattenhoff
et al., 2013), bvhtdAGIL did not affect expression of pluripotency
markers such as Oct4 and Nanog, and mutant ESCs showed
normal morphology and self-renewal properties as well as typical
cell-cycle kinetics (Figures 2E, 2F, and S2B). We then tested
whether bvhtdAGIL could form embryoid bodies (EBs), which
give rise to derivatives of all three germ layers. Notably, CMs
can form in EBs and can be visualized as beating cell clusters.
We allowed WT and mutant ESCs to aggregate in the absence
of pluripotency growth factors and then measured the percent-
age of spontaneously beating EBs at different time points. We
found that bvht dAGIL EBs show significantly reduced beating
( 5%) compared to WT cells ( 25%) at day 10, similar to our ob-
servations in Bvht-depleted EBs (Klattenhoff et al., 2013).
Helical junctions are often important for the structural and cata-
lytic properties of RNAs (Bindewald et al., 2008). For example, a
four-way junction promotes the functional folded state of the
hairpin ribozyme (Tan et al., 2003). Thus, we also introduced
mismatch mutations into the H4 region ( bvhtH4mis) to destabilize
the 5WJ by CRISPR/Cas9-mediated HDR and selected clones
(Figures S2C–S2E). In contrast to bvhtdAGIL, alteration of the H4 re-
gion did not significantly affect the percentage of beatingEBs (Fig-
ure 2G). BvhtdAGIL EBs also displayed a failure to activate genes
associated with the cardiac contractile apparatus such as cardiac
troponin T (cTnT) and myosin heavy chain genes, whereas
bvhtH4mis EBs showed normal expression comparable toWT con-
trols (Figure 2H). In contrast, neuronal and endodermal genes
were expressed normally in bvhtdAGIL EBs in response to retinoic
acid treatment similar to WT and bvhtH4mis EBs (Figures S2F and
S2G). Although these data do not preclude a secondary role for
the 5WJ, our analysis suggests that the Bvht AGILmotif is required
for formation of spontaneously contracting EBs.
Braveheart AGIL Motif Is Necessary for Cardiovascular
Lineage Commitment
To further dissect AGIL function in the cardiovascular lineage, we
employed a directed in vitro CM differentiation assay that per-
mits isolation of cell populations at well-defined stages (ESCs,
precardiac mesoderm [MES], CPs, and CMs) (Kattman et al.,
2011; Wamstad et al., 2012) (Figure 3A). At each stage, cells
are subject to fluorescence-activated cell sorting (FACS) using
antibodies against specific markers to quantify differentiation
efficiency. Using this approach, we routinely isolate a high
percentage of Pdgfra+, Flk1+ (MES), Nkx2.5-GFP+ (CP), and
cTnT+ (CM) cell populations (Figure 3B). In contrast, FACS of
bvhtdAGIL cells showed a striking reduction in the percentage of
CP and CM marked cells during differentiation. We also demon-
strate that although bvhtdAGIL and WT cells showed similar
morphology at day 4 (MES), immunofluorescence of the cultures
at day 5.3 (CP) and day 10 (CM) using antibodies against Nkx2.5-
GFP or cTnT, respectively, showed no staining in the mutant
cells (Figure 3C). These results are highly reproducible among
multiple independent bvhtdAGIL ESC clones and similar to shRNA
depletion of Bvht (Figures S3A, S3B, and S3E), suggesting that
the differentiation defects are not due to off-target effects.
We next analyzed the expression of a set of cardiac TFs
that failed to activate upon shRNA-mediated depletion of
Bvht (Figures S3C and S3D) (Klattenhoff et al., 2013). The meso-
dermal marker Brachyury showed higher expression at day 4 in
bvhtdAGIL cells and sustained expression at day 5.3 compared to
WT controls (Figure 3D). MesP1 is one of the earliest known
markers of a common multi-potent cardiovascular progenitor
(Bondue et al., 2008; Lindsley et al., 2008) and showed
decreased expression at day 4 (MES) in bvht -shRNA-depleted
cells (Klattenhoff et al., 2013). Although MesP1 expression
showed no change in the bvhtdAGIL mutant, we observed a failure
to activate the cardiac TFs downstream of this factor, including
Nkx2.5, Gata4, Gata6, Hand1, Hand2, Tbx5, and Mef2c,
compared to WT cells, suggesting that distinct regions of Bvht
contribute to its total activity (Figure 3E). These data are highly
reproducible using multiple independent ESC clones (Fig-
ure S3F). Moreover, expression of WT Bvht from the ROSA26
locus in the bvhtdAGIL background (Figure S3G) rescued the
CM differentiation defect, indicating that the phenotype is due
to loss of AGIL function (Figures S3H–S3K). Together, our data
point to a central role for the Bvht AGIL motif in specifying the
cardiovascular lineage.
Braveheart AGIL Interacts with Factors that Bind G-Rich
Nucleic Acids
A prevailing model suggests that lncRNAs act as molecular scaf-
folds, mediating interactions with proteins (Geisler and Coller,
2013; Quinn and Chang, 2016; Rinn and Chang, 2012). Although
genome-wide studies support binding between lncRNAs and
proteins, few studies have identified RNA structural motifs
(D) Northern blot analysis showing the levels of Bvht transcripts in indicated ESC lines. rRNAs are used for loading control.
(E) qRT-PCR analysis showing the levels of Bvht and ESC pluripotency markers Oct4 and Nanog in indicated ESC lines. Experiments were performed in triplicate
and data are represented as mean values ± SD.
(F) Immunofluorescence staining of indicated ESCs using Oct4 antibody. Nuclei were stained with DAPI. BF, bright field. Scale bar, 100 m m.
(G) Percentage of spontaneously contracting embryoid bodies (EBs) at day 12 of differentiation (n > 200) from indicated ESCs.
(H) qRT-PCR analysis of EBs at day 12 showing the relative levels of CM markers from indicated ESC lines.
All experiments were performed in triplicate and data are represented as mean values ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed Student’s
t test).
See also Figure S2.
Molecular Cell 64 , 37–50, October 6, 2016 41