Figure 3
Rapid and efficient TSC2 gene inactivation by CRISPR/Cas9 gene inactivation in iCas9- hPSC-derived neurons.
(a) Strategy to obtain an enriched population of TSC2-edited neurons from iCas9-hPSCs. The differentiation protocol, based on the forced expression of Ngn2, is coupled to TSC2 gene mutagenesis when the sgRNA-T4 guide is also transduced. Doxycycline (added at DIV-1) and blasticidin (DIV 3) were maintained for all the experiment. Representative FACS plots of EGFP levels in iCas9-hPSCs. The lentiviruses used to infect each sample were: Ngn2-blast for the negative control, Ngn2-GFP-blast for the control and Ngn2-GFP-blast-sgRNA-T4 for the sgRNA-T4 sample. The EGFP population selected is enclosed in the box. (b) RFLP assay performed in control sorted, sgRNA-T4 bulk and sgRNA-T4 sorted populations using the SfcI enzyme. Middle, representative TIDE assay for either the sgRNA-T4 bulk or sgRNA-T4 sorted populations. Right, indel quantification by RFLP and TIDE in sgRNA-T4 bulk and sorted cells (n = 2). Number of wild type (wt), frameshift mutations (mut fs) and non frameshift mutations (mut nfs) found among 40 sequences analyzed in sgRNA-T4 sorted cells. (c) Western-blots to detect TSC2, PS6, S6 and Calnexin (CNX) protein levels in control and sgRNA-T4 neurons. Densitometric quantification of TSC2, PS6 and S6 relative to CNX or S6 protein levels in arbitrary units (n = 4 for TSC2, n = 5 for the others, *p < 0.05). (d) Immunostaining of the sorted cells for MAP2 and PS6. Quantification of PS6/MAP2 double positive cells respect to the total MAP2-positive cell population (n = 3, *p < 0.05). Nuclei were stained with Hoechst. Scale bar: 20 μm. Full-length gel and blots are included in Figure S7c and d.
