## 2 Results
### Characterizing the Pluripotency and Stability of G56R-CRISPR iPSC Lines  
Afterwards, we assayed whether the G56R-CRISPR2 and -3 lines retained their iPSC characteristics and genetic stability, in comparison to the parental G56R iPSC line. By qPCR analysis, we detected the expression of the pluripotency genes _NANOG_, _OCT3/4_, and _LIN28A_ in the G56R-CRISPR2 and -3 lines at similar levels to the parental G56R line (Figure 4A) and, by IF analysis, the expression of the pluripotency markers NANOG, OCT3/4, and SOX2 (Figure 4B). In addition, the G56R-CRISPR2 and -3 lines differentiated into the three germ layers, as determined by IF analysis of AFP expression for endoderm, SMA for mesoderm, and glial fibrillary acidic protein (GFAP) for ectoderm (Figure 4C). Moreover, we tested genetic stability of the G56R-CRISPR2 and -3 iPSC lines by a digital PCR test of the copy number variant (CNV) of the most commonly rearranged chromosomal regions reported in iPSCs [34]. A CNV of 2 was detected for the autosomes, and a CNV of 1 for the X chromosome, in both lines indicating that the G56R-CRISPR2 and -3 iPSCs were genetically stable (Figure 4D).  
Figure 3: Generation of G56R-CRISPR iPSC lines. (**A**) Representative electropherogram of the sequence flanking the DSB site in the DNA of the G56R-CRISPR3 and -4 iPSC clones. The sequence begins at the c.166G>A mutational site. The introduced nucleotide (c.174 insA) is boxed in red. (**B**) Alignment of the Sanger sequencing results for the same region shown in (**A**) in the wild type, G56R and four G56R-CRISPR IPSC clones. The mutational site is shown in red. The indels are indicated in blue. The PAM sequence is shown in green. (**C**) Graphical representation of the protein structure of 410 aa WT NR253 (left) with the DNA binding domain (DBD) shown in blue, the ligand binding domain (LBD) shown in green and the N-terminus and the hinge region are shown in grey. The predicted effect on NR253 (right) following introduction of the frameshift mutation on the G56R (indicated by a red star in the truncated DBD) mutant allele. The red bar indicates a non-NR2E3 protein sequence prior to the premature termination at 139 aa.  
Finally, we evaluated potential off-target events that may have been introduced in the G56R-CRISPR clones. We PCR-amplified the off-target sites for gRNA1 predicted by the  
Figure 4: Characterization of the G56R-CRISPR2 and -3 iPSC lines. (**A**) qPCR analysis of the expression of the host pluripotency genes _NANOG_ (left), _OCT3/4_ (middle), and _LIN28A_ (right) in cDNA from G56R and G56R-CRISPR2 and -3 iPSCs. Results are expressed as mean \(\pm\) SEM (\(n\) = 3). (**B**) Representative images of IF analysis of the expression of endogenous NANOG (left; G56R-CRISPR3), OCT3/4 (middle; G56R-CRISPR2), and SOX2 (right; G56R-CRISPR2) in G56R-CRISPR2 and -3 iPSCs; nuclei labelled in blue. (**C**) Representative images of IF analysis following embryoid body differentiation of G56R-CRISPR2 and -3 iPSCs for the expression of the germ layer markers AFP (endoderm; left; G56R-CRISPR3), SMA (mesoderm; middle; G56R-CRISPR3), and glial fibrillary acidic protein (GFAP) (ectoderm; right; G56R-CRISPR2). (**D**) Representative digital qPCR analysis of the G56R-CRISPR3 iPSC line for the presence of the most commonly rearranged chromosomal regions detected in iPSCs. A copy number of \(\sim\)2 for the autosomes, and 1 for the sex chromosomes, is indicative of stability.  
CRISPRR software from the DNA of the G56R-CRISPRR2 and -3 lines while using specific primers and analyzed the amplicons by Sanger sequencing; to have a complete overview of potential off-target events, we additionally tested G56R-CRISPRR4. The sequences for each of these sites in the G56R-CRISPRR2, -3 and -4 clones were identical to that of WT, demonstrating the absence of off-target events, as shown in Supplementary Figure S2.  
In conclusion, following eSpCas9-mediated genome-editing, the resulting G56R-CRISPR iPSC lines retained their pluripotency and genetic stability, and they did not harbor off-target events.