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CRISPR/Cas9 genome editing is a rapidly advancing technology that has the potential to accelerate research and development in a variety of fields. However, manual genome editing processes suffer from limitations in scalability, efficiency, and standardization. The implementation of automated systems for genome editing addresses these challenges, allowing researchers to cover the increasing need and perform large-scale studies for disease modeling, drug development, and personalized medicine. In this study, we developed an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. We implemented a 4D-Nucleofector with a 96-well shuttle device into the StemCellFactory, optimized several parameters for single cell culturing and established an automated workflow for CRISPR/Cas9-based genome editing. When validated with a variety of genetic backgrounds and target genes, the automated workflow showed genome editing efficiencies similar to manual methods, with indel rates of up to 98%. Monoclonal colony growth was achieved and monitored using the StemCellFactory-integrated CellCelector, which allowed the exclusion of colonies derived from multiple cells or growing too close to neighbouring colonies. In summary, we demonstrate the successful establishment of an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. The development of such a standardized and scalable automated CRISPR/Cas9 system represents an exciting new tool in genome editing, enhancing our ability to address a wide range of scientific questions in disease modeling, drug development and personalized medicine.

Induced pluripotent stem cells (iPSCs) and their derivatives have been described to display epigenetic memory of their founder cells, as well as de novo reprogramming-associated alterations. In order to selectively explore changes due to the reprogramming process and not to heterologous somatic memory, we devised a circular reprogramming approach where somatic stem cells are used to generate iPSCs, which are subsequently re-differentiated into their original fate. As somatic founder cells, we employed human embryonic stem cell-derived neural stem cells (NSCs) and compared them to iPSC-derived NSCs derived thereof. Global transcription profiling of this isogenic circular system revealed remarkably similar transcriptomes of both NSC populations, with the exception of 36 transcripts. Amongst these we detected a disproportionately large fraction of X chromosomal genes, all of which were upregulated in iPSC-NSCs. Concurrently, we detected differential methylation of X chromosomal sites spatially coinciding with regions harboring differentially expressed genes. While our data point to a pronounced overall reinstallation of autosomal transcriptomic and methylation signatures when a defined somatic lineage is propagated through pluripotency, they also indicate that X chromosomal genes may partially escape this reinstallation process. Considering the broad application of iPSCs in disease modeling and regenerative approaches, such reprogramming-associated alterations in X chromosomal gene expression and DNA methylation deserve particular attention.