A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Reto Eggenschwiler - , Leibniz University Hannover (LUH) (Author)
  • Thomas Gschwendtberger - , Leibniz University Hannover (LUH) (Author)
  • Christian Felski - , Leibniz University Hannover (LUH) (Author)
  • Christopher Jahn - , Leibniz University Hannover (LUH) (Author)
  • Florian Langer - , Leibniz University Hannover (LUH) (Author)
  • Jared Sterneckert - , Chair of iPS Cells and Neurodegenerative Diseases (Author)
  • Andreas Hermann - , University of Rostock, German Center for Neurodegenerative Diseases (DZNE) (Author)
  • Jonathan Lühmann - , Leibniz University Hannover (LUH) (Author)
  • Doris Steinemann - , Leibniz University Hannover (LUH) (Author)
  • Alexandra Haase - , Leibniz University Hannover (LUH), German Center for Lung Research (DZL) (Author)
  • Ulrich Martin - , Leibniz University Hannover (LUH), German Center for Lung Research (DZL) (Author)
  • Susanne Petri - , Leibniz University Hannover (LUH) (Author)
  • Tobias Cantz - , Leibniz University Hannover (LUH), Max Planck Institute for Molecular Biomedicine (Author)

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.

Details

Original languageEnglish
Article number22154
JournalScientific reports
Volume11
Issue number1
Publication statusPublished - 12 Nov 2021
Peer-reviewedYes

External IDs

PubMed 34773059
ORCID /0000-0002-7688-3124/work/142250009