Figure 3.

KD of Cyrano with CRISPRi Approach Has No Impact on the Pluripotency of Human iPSCs

(A) Schematic overview of the generation of inducible Cyrano KD human iPSCs using a CRISPRi approach.

(B) Representative RNA FISH images stained with a Cyrano-specific probe in unmodified cells (CRISPRi) and one clone, with and without doxycycline (doxy) treatment. Nuclei were stained with DAPI. Scale bar, 5 μm.

(C) Expression of Cyrano, NANOG, and SOX2 after doxy treatment in the monoclonal population. Expression levels were measured by qRT-PCR, mean ± SD of three independent experiments are shown, unpaired t test was performed for statistical analysis.

(D) Representative images of alkaline phosphatase staining of one KD clone with and without doxy treatment. Scale bar, 500 μm.

(E) Flow cytometry analysis of SSEA4 and TRA1-60 of one clone. Isotype controls (light gray, filled) and unstained cells (dark gray line) were used as controls. Representative plots with mean ± SD of three independent experiments are depicted.

(F) Fluorescence immunocytochemistry of NANOG, OCT4, and SOX2 was performed in Cyrano KD cells after doxy treatment. Scale bar, 100 μm.

(G) Expression levels were measured by qRT-PCR, mean ± SD of three independent experiments, unpaired t test was performed for statistical analysis.

(H) miR-7-5p was overexpressed in CRISPRi cells. Expression levels were measured by qRT-PCR, mean ± SD of three independent experiments, unpaired t test was performed for statistical analysis.

(I) Volcano plot depicting the results of the RNA sequencing of Cyrano KD clone with and without doxy treatment of three experiments. Cut off criteria −1 < log2(FC) > 1, q > 2. IPA network analysis.

(J) RNA-seq tracks of CRISPRi 1II E12 with and without doxy.

(K) Normalized read counts using DEseq2 of pluripotency-related and Cyrano-related genes.