A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

Article Article scientifique Article A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming eng 2019-11-28 2020-01-10 author Kesselring Lisa Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Miskey Csaba Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Zuliani Cecilia 0000-0001-6026-7207 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany author Querques Irma 0000-0003-1113-2773 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany 419568 author Kapitonov Vladimir Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Laukó Andrea BioTalentum Ltd, Gödöllő, 2100 Gödöllő, Hungary author Feher Anita BioTalentum Ltd, Gödöllő, 2100 Gödöllő, Hungary 813578 author Palazzo Antonio 0000-0003-0961-6832 Department of Biology, University of Bari ‘Aldo Moro’, Italy author Diem Tanja Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Lustig Janna Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Sebe Attila 0000-0001-7814-5144 Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany author Wang Yongming 0000-0001-8269-5296 Mobile DNA, Max Delbrück Center for Molecular Medicine, Berlin, Germany author Dinnyés András BioTalentum Ltd, Gödöllő, 2100 Gödöllő, Hungary author Izsvák Zsuzsanna Mobile DNA, Max Delbrück Center for Molecular Medicine, Berlin, Germany author Barabas Orsolya 0000-0002-2873-5872 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany 938832 Département de biologie moléculaire et cellulaire (MOCEL) author Ivics Zoltán 0000-0002-7803-6658 Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany 316-331 10.1093/nar/gkz1119 31777924 0305-1048 PMC6943129 https://academic.oup.com/nar/article/48/1/316/5644994 The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int− transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int− transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change. European Commission Comparative stem cell research in mouse and humans 230675 INDUSTEM EU FP7 European Molecular Biology Laboratory true Nucleic acids research 48 1 d07b5b1e-96af-4827-b767-1915c8e79da0 Article (Published version) Public Article-scientifique-from-unpaywall.pdf 4162454 application/pdf CC BY-NC-4.0 1210c72822f2e02eac7447c0be5c516e35dab984c6ebc26177ab6e12795c38ef openAccess

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A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming	Nucleic acids research			2020	105	72

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