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@ARTICLE{Eggenschwiler:163639,
      author       = {Eggenschwiler, Reto and Gschwendtberger, Thomas and Felski,
                      Christian and Jahn, Christopher and Langer, Florian and
                      Sterneckert, Jared and Hermann, Andreas and Lühmann,
                      Jonathan and Steinemann, Doris and Haase, Alexandra and
                      Martin, Ulrich and Petri, Susanne and Cantz, Tobias},
      title        = {{A} selectable all-in-one {CRISPR} prime editing piggy{B}ac
                      transposon allows for highly efficient gene editing in human
                      cell lines.},
      journal      = {Scientific reports},
      volume       = {11},
      number       = {1},
      issn         = {2045-2322},
      address      = {[London]},
      publisher    = {Macmillan Publishers Limited, part of Springer Nature},
      reportid     = {DZNE-2022-00385},
      pages        = {22154},
      year         = {2021},
      abstract     = {CRISPR prime-editors are emergent tools for genome editing
                      and offer a versatile alternative approach to HDR-based
                      genome engineering or DNA base-editors. However, sufficient
                      prime-editor expression levels and availability of optimized
                      transfection protocols may affect editing efficiencies,
                      especially in hard-to-transfect cells like hiPSC. Here, we
                      show that piggyBac prime-editing (PB-PE) allows for
                      sustained expression of prime-editors. We demonstrate
                      proof-of-concept for PB-PE in a newly designed lentiviral
                      traffic light reporter, which allows for estimation of gene
                      correction and defective editing resulting in indels, based
                      on expression of two different fluorophores. PB-PE can
                      prime-edit more than $50\%$ of hiPSC cells after antibiotic
                      selection. We also show that improper design of pegRNA
                      cannot simply be overcome by extended expression, but PB-PE
                      allows for estimation of effectiveness of selected pegRNAs
                      after few days of cultivation time. Finally, we implemented
                      PB-PE for efficient editing of an amyotrophic lateral
                      sclerosis-associated mutation in the SOD1-gene of
                      patient-derived hiPSC. Progress of genome editing can be
                      monitored by Sanger-sequencing, whereas PB-PE vectors can be
                      removed after editing and excised cells can be enriched by
                      fialuridine selection. Together, we present an efficient
                      prime-editing toolbox, which can be robustly used in a
                      variety of cell lines even when non-optimized
                      transfection-protocols are applied.},
      keywords     = {Amyotrophic Lateral Sclerosis: genetics / CRISPR-Cas
                      Systems / Cell Line / Gene Editing: methods / HEK293 Cells /
                      Humans / Induced Pluripotent Stem Cells: metabolism /
                      Mutation / Superoxide Dismutase-1: genetics / Transfection:
                      methods / Superoxide Dismutase-1 (NLM Chemicals)},
      cin          = {AG Teipel},
      ddc          = {600},
      cid          = {I:(DE-2719)1510100},
      pnm          = {353 - Clinical and Health Care Research (POF4-353)},
      pid          = {G:(DE-HGF)POF4-353},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:34773059},
      pmc          = {pmc:PMC8589839},
      doi          = {10.1038/s41598-021-01689-2},
      url          = {https://pub.dzne.de/record/163639},
}