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Directed Evolution of Reprogramming Factors by Cell Selection and Sequencing.

ABSTRACT: Directed biomolecular evolution is widely used to tailor and enhance enzymes, fluorescent proteins, and antibodies but has hitherto not been applied in the reprogramming of mammalian cells. Here, we describe a method termed directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) to identify artificially enhanced and evolved reprogramming transcription factors. DERBY-seq entails pooled screens with libraries of positionally randomised genes, cell selection based on phenotypic readouts, and genotyping by amplicon sequencing for candidate identification. We benchmark this approach using pluripotency reprogramming with libraries based on the reprogramming factor SOX2 and the reprogramming incompetent endodermal factor SOX17. We identified several SOX2 variants outperforming the wild-type protein in three- and four-factor cocktails. The most effective variants were discovered from the SOX17 library, demonstrating that this factor can be converted into a highly potent inducer of pluripotency with a range of diverse modifications. We propose DERBY-seq as a broad-based approach to discover reprogramming factors for any donor/target cell combination applicable to direct lineage reprogramming in vitro and in vivo.

SUBMITTER: Veerapandian V

PROVIDER: S-EPMC6092915 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Directed Evolution of Reprogramming Factors by Cell Selection and Sequencing.

Veerapandian Veeramohan V Veerapandian Veeramohan V Ackermann Jan Ole JO Srivastava Yogesh Y Malik Vikas V Weng Mingxi M Yang Xiaoxiao X Jauch Ralf R

Stem cell reports 20180802 2

Directed biomolecular evolution is widely used to tailor and enhance enzymes, fluorescent proteins, and antibodies but has hitherto not been applied in the reprogramming of mammalian cells. Here, we describe a method termed directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) to identify artificially enhanced and evolved reprogramming transcription factors. DERBY-seq entails pooled screens with libraries of positionally randomised genes, cell selection based o ...[more]

PMID: 30078555

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Project description:Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acid (XNA) synthetases and reverse transcriptases capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

Dataset Information

Directed Evolution of Reprogramming Factors by Cell Selection and Sequencing.

Publications

Directed Evolution of Reprogramming Factors by Cell Selection and Sequencing.

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