Targeting plasmids (exon 8: TALENs (3 ug each) and pCAG-GFP (1 ug) or exon 2: pCAG-Cas9-T2A-GFP (2.5 ug) and gRNA (2.5 ug)) were electroporated into dissociated YZ1 iPSC line (~5E6 cells, dissociated in Accutase (Life Technologies) supplemented with 10 uM ROCK inhibitor, Y-27632(Stem RD)) using the Amaxa 4D-Nucleofector X Unit (Lonza, program DN-100). Transfected cells were plated onto Matrigel- (BD Biosciences) coated plates in mTeSR1 media (Stemcell Technologies) + 10 uM ROCK inhibitor. 48 hours after transfection, iPSCs were dissociated and GFP+ cells were collected by FACS. GFP+ cells were plated at low density onto irradiated MEFs (GFP+ cells: ~3000 cells/cm2, MEFs: ~17500 cells/cm2, GlobalStem). Cells were cultured in standard iPS cell medium until small, isolated colonies appeared (~7-10 days). Clones were transferred to individual wells of a matrigel-coated 96 well plate and expanded in mTeSR1 media. Genomic DNA was harvested using QuickExtract DNA Extraction solution (Epicentre). Clones were screened for genomic mutation by PCR amplification around the target site, followed by Sanger sequencing.
Neural aggregates (~day 27-31) were dissociated using Accutase + 10 uM ROCK Inhibitor. Cells were resuspended in in P3 solution (Lonza) and DNA was added (3 ug Super8XTOPFlash, 1.5 ug pMIR-REPORT-beta-galactosidase,1-5E6 cells). Cells were electroporated by the Amaxa 4D-Nucleofector X Unit (Lonza, program CU-133). Electroporated cells were recovered in RPMI + 10% B27 supplement + 20 uM ROCK Inhibitor for 15 minutes and subsequently plated onto poly-ornithine/laminin coated 96 well plates (4 ug/cm2 poly-ornithine, 1 ug/cm2 laminin, coated overnight at 37°C) containing N2/B27 neural induction media + 10 uM ROCK-Inhibitor. 16 hours later, media was changed for 24 hours. Cells were then lysed in luciferase assay lysis buffer (Biotium) and used for a firefly luciferase assay (Biotium) and beta-galactosidase assay (Promega).
The authors used Nucleofector 4D in combination with TALEN and CRISPER-CAS9 technologies for the genome editing of hiPSC lines, generating disease-relevant gene disruption in DISC1, a gene linked with major mental illness. This affected neural progenitor cell proliferation, baseline WNT signaling, and the expression of Neural Precursor Cells fate markers such as FoxG1 and Tbr2. This supports the hypothesis that DISC1-dependent suppression of basal Wnt signaling influences the distribution of cell types engendered during cortical development.