GSE107987
Directed evolution of reprogramming factors by cell selection and sequencing
Directed biomolecular evolution is widely used to tailor and enhance proteins but has hitherto not been applied in the reprogramming of mammalian cells. Here we describe a novel method to identify artificially enhanced and evolved reprogramming factors by pooled screens with randomised protein libraries, cell selection based on phenotypic readouts and genotyping by amplicon sequencing. We benchmark this approach by identifying artificially enhanced Sox2 and Sox17 factors in pluripotency reprogramming. Overall design: Mouse embryonic fibroblasts with a transgenic Oct4-GFP reporter (OG2-MEFs) were reprogrammed with Sox2,Oct4, Klf4 and c-Myc under Serum/LIF/Vitamin C conditions. Directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) screens were performed by replacing Sox2 with eSox2 and eSox17 libraries. In the libraries three amino acids in the DNA binding HMG box were subjected to NNK randomisation. Cells were sorted by FACS and eSOX factors in GFP positive and GFP negative cell populations were genotyped by amplicon sequencing to identify variants with improved reprogramming activity.  Libraries were prepared by PCR amplifying genomically integrated retroviral transgenes. Sequencing adaptors and barcodes were added during the PCR. Three independent biological replicates and two technical replicates (PCR and library prep ) were done for eSox2 DERBY-seq experiments. Three independent biological replicates were performed for eSox17 DERBY-seq experiments. Input pMX plasmid libraries were sequenced for both DERBY-seq experiments respectively. We performed ChIP sequencing of wildtype Sox17 and an eSox17 mutant at  reprogramming  days 3 and 6. RNA sequencing was performed for Sox17, Sox2, eSox2,eSox17 and GFP conditions at day 3, 6, 9 and iPSCs. >>>Submitter states that they will update the GEO records with the nature of the mutants upon publication.<<<