Enhancing cellular reprogramming by directed factor evolution

Abstract

To decode sequence-function relationship of transcription factor (TF) mediated cell fate conversions we use structural modeling and quantitative biochemical assays to analyze the formation of TF complexes on regulatory DNA in combination with genomics techniques. By contrasting the dynamic binding and gene regulation of paralogous TFs and engineered factors we begin to appreciate ‘enhancer codes’ directing cell fate programming. Further, we could identify functionally critical structural elements endowing selected TF to direct this process. We specifically ask how combinations of lineage specifying TFs (including SOX, OCT, PAX and FOX family proteins) work together to guide cell fate conversions in a step-wise manner. Lessons learned from these studies led us to hypothesize that native TFs are not optimized to direct artificial cell fate conversions and that they can be enhanced by directed evolution. To test this concept we generate TF libraries by randomizing selected amino acids and by recombining domains of paralogous genes. Using these libraries we perform pooled library screens, cell selection based on phenotypic read-outs and amplicon sequencing. This way, we identify artificially evolved TFs (eTFs) that program cell fates faster, more efficiently and in a more controlled fashion outperforming their wild-type counterparts. We propose that artificial reprogramming factor evolution presents a general paradigm that can be applied to any biomolecule-driven cell conversion system with utility in regenerative biomedicine.