DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes [RNA-Seq]

We generated iPS cells knock out (KO) for two Aicardi Goutières syndrome genes, TREX1 and RNASEH2b to model the disease in a human neurological context. Their transcriptomes were analyzed at several differentiation stages: iPSC-derived Neural Stem Cells, iPSC-derived proinflammatory astrocytes and iPSC-derived neurons. We then evaluated when and to which extent the AGS-related transcriptional alterations arised in all KO and WT cells. To study AGS pathology in a physiologically relevant neural context we generated isogenic iPSCs knock-out (KO) for TREX1 or RNASEH2B. Healthy donor-derived iPS cells were engineered through the CRISPR-Cas9 technology. All editing reagents were delivered transiently through electroporation, followed by limiting dilution of single clones of which we isolated around 30-40 per condition for molecular screening. All clones were analyzed by mismatch-sensitive molecular assay, Sanger sequencing of individual alleles obtained through TOPO TA cloning assay and by Western Blot (WB). One bi-allelic KO clone per gene was selected. A clone obtained from the screen harboring the WT genome was selected as a WT reference for all further experiments. At first, WT, T-/- and R-/- clones were differentiated into induced neural stem/progenitor cells (iPSC-NSCs) (Chambers et al., 2009), Later they were differentiated in more committed cells such inflammation-responsive astrocytes as previously reported (Santos et al., 2017) and towards neurons following a commercial differentiation protocol. Pluripotent stem cells and more committed derived cells were analyzed by RNA Seq to characterize the transcriptional changes at different stages of differentiations.