Fig. 3

Quantitative analysis of CREB expression in the CRISPR/Cas9-transfected cortical neurons. Cultured cortical neurons were transfected with pTα1-EGFP (control, a–d) or the CRISPR/Cas9 vector together with pTα1-EGFP (CRISPR/Cas9, e–h). The cultures were fixed after 6 days following the transfection. ICC analysis was performed with multi-color fluorescence labeling of EGFP (Alexa488, green a, e), CREB (Cy3, red b, f) and DAPI (blue c, g). Scale bar 10 μm. i Scatter diagram shows the immunofluorescence intensities of EGFP and CREB expression in the neurons co-transfected with CRISPR/Cas9 vector and pTα1-EGFP (CRISPR/Cas9, black) and pTα1-EGFP transfected neurons (control, white) at 7 DIV. A red circle indicates the neurons with strong EGFP and weak CREB expression. j Distribution histogram shows the CREB expression level in the CRISPR/Cas9-transfected neurons (black) and the control neurons (white) at 7 DIV. The difference between the two distributions is significant (KS test, P < 0.001). Interrupted vertical lines indicate boundaries of the estimated fractions which correspond to the very low and the moderate expression of CREB in the CRISPR/Cas9-transfected neurons. k Representative mutation patterns of the target site in the exon 7 of Creb revealed by single-cell genome PCR and DNA sequencing in the CRISPR/Cas9-transfected neurons. ICC analysis was performed with anti-CREB antibody (left side images). Top indicates the targeted sequence (blue underline) and PAM sequence (red underline) in wild-type Creb locus. Red dots indicate deleted bases. The number of deletions (−) and insertions (+) are shown. l Histogram shows the frequency of the homozygous mutant neurons in the CRISPR/Cas9-transfected neurons in different culture periods. (4 DIV, n = 36 cells; 7 DIV, n = 69 cells; 10 DIV, n = 53 cells)