Journal Article

DZNE-2022-00385

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.

Eggenschwiler, R. ; Gschwendtberger, T. ; Felski, C. ; Jahn, C. ; Langer, F. ; Sterneckert, J. ; Hermann, A.DZNE* ; Lühmann, J. ; Steinemann, D. ; Haase, A. ; Martin, U. ; Petri, S. ; Cantz, T.

2021
Macmillan Publishers Limited, part of Springer Nature [London]

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Please use a persistent id in citations: doi:10.1038/s41598-021-01689-2

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.

Keyword(s): Amyotrophic Lateral Sclerosis: genetics (MeSH) ; CRISPR-Cas Systems (MeSH) ; Cell Line (MeSH) ; Gene Editing: methods (MeSH) ; HEK293 Cells (MeSH) ; Humans (MeSH) ; Induced Pluripotent Stem Cells: metabolism (MeSH) ; Mutation (MeSH) ; Superoxide Dismutase-1: genetics (MeSH) ; Transfection: methods (MeSH) ; Superoxide Dismutase-1

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Contributing Institute(s):

1. Clinical Dementia Research Rostock /Greifswald (AG Teipel)

Research Program(s):

1. 353 - Clinical and Health Care Research (POF4-353) (POF4-353)

Appears in the scientific report 2021

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