DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes [DNA-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. However, it has been reported that after precise genome engineering human iPSCs may show severe alterations of the p53 gene. With the aim to evaluate the genome engeneering impact on the p53 pathway in the CRISPR-CAS9 knock out clones we verified integrity of the p53 genome sequence after gene editing. Overall design: 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. To check if Trex1 and RNaseH2b knock out iPSCs showed any mutation on p53 gene we isolated them from their murine embrionic fibroblasts feeder cells. Avoiding any murine DNA contamination we isolated the human iPSCs genomic DNA sequencing for p53. We then compared the KO sequences to the WT one. WT samples came from a negative clone.