Fig. 6

CRISPR-dCas9 mediated gene activation/repression in iMG. A & B. Schematic for drug-inducible (tetracycline/doxycycline) dCas9-VP64 (activation) and dCas9-KRAB (repressor) expression vectors, respectively. Target sgRNAs were expressed with Tet-dCas9-VP64 or Tet-dCas9-KRAB domains at iPSCs stages, while doxycycline was added at day 16 of differentiation. C. Quantitative analysis of mRNA expression of SORL1 by RT-qPCR, after lentiviral expression of control (C) and SORL1 promoter (S) targeting sgRNAs in iPSCs with stably integrated constitutive dCas9-VP64 (mean ± SD, n = 3, t test P = 0.0032). D. Functional validation of doxycycline-inducible dCas9-VP64 (CRISPRa) via quantitative PCR on SORL1 mRNA levels in iMG expressing SORL1 promoter targeting sgRNA or control sgRNA (mean ± SD, n = 3, ANOVA, P < 0.0001). Doxycycline was added on day 16 of differentiation. E. Immunoblotting demonstrates overexpression of SOLR1 depends on doxycycline treatment after lentiviral deliveries of Tet-dCas9-VP64 and sgRNA targeting SORL1 promoter (SORL1(p)). (Con. dC9-VP-64 stands for: constitutive expression of dCas9-VP64). The blot is representative of three independent experiments. Complete western blot gel is provided in supplementary Figure S7. F. RT-qPCR analysis of mRNA expression of SORL1, after lentiviral expression of control (C) and SORL1 promoter (S) targeting sgRNAs in iPSCs with stably integrated constitutive dCas9-KRAB domain (P < 0.05, (mean ± SD, n = 3, t test P = 0.01). G. Drug-inducible dCas9-KRAB (CRISPRi) represses SORL1 transcription only when doxycycline is added to the cell culture. The bar chart shows the quantitative difference in mRNA levels, measured by RT-qPCR, in iMG expressing SORL1 promoter targeting sgRNA or control sgRNA (mean ± SD, n = 3, ANOVA, P < 0.0001). Doxycycline was added on day 16 of differentiation. H. G-banded chromosome analysis shows a normal karyotype of differentiated iMG