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| Home > Publications Database > A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines. > print |
| Home > Publications Database > A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines. > print |
| 001 | 163639 | ||
| 005 | 20230915092425.0 | ||
| 024 | 7 | _ | |a 10.1038/s41598-021-01689-2 |2 doi |
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| 037 | _ | _ | |a DZNE-2022-00385 |
| 041 | _ | _ | |a English |
| 082 | _ | _ | |a 600 |
| 100 | 1 | _ | |a Eggenschwiler, Reto |0 0000-0003-3588-7847 |b 0 |
| 245 | _ | _ | |a A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines. |
| 260 | _ | _ | |a [London] |c 2021 |b Macmillan Publishers Limited, part of Springer Nature |
| 336 | 7 | _ | |a article |2 DRIVER |
| 336 | 7 | _ | |a Output Types/Journal article |2 DataCite |
| 336 | 7 | _ | |a Journal Article |b journal |m journal |0 PUB:(DE-HGF)16 |s 1655197285_926 |2 PUB:(DE-HGF) |
| 336 | 7 | _ | |a ARTICLE |2 BibTeX |
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| 336 | 7 | _ | |a Journal Article |0 0 |2 EndNote |
| 520 | _ | _ | |a 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. |
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| 650 | _ | 7 | |a Superoxide Dismutase-1 |0 EC 1.15.1.1 |2 NLM Chemicals |
| 650 | _ | 2 | |a Amyotrophic Lateral Sclerosis: genetics |2 MeSH |
| 650 | _ | 2 | |a CRISPR-Cas Systems |2 MeSH |
| 650 | _ | 2 | |a Cell Line |2 MeSH |
| 650 | _ | 2 | |a Gene Editing: methods |2 MeSH |
| 650 | _ | 2 | |a HEK293 Cells |2 MeSH |
| 650 | _ | 2 | |a Humans |2 MeSH |
| 650 | _ | 2 | |a Induced Pluripotent Stem Cells: metabolism |2 MeSH |
| 650 | _ | 2 | |a Mutation |2 MeSH |
| 650 | _ | 2 | |a Superoxide Dismutase-1: genetics |2 MeSH |
| 650 | _ | 2 | |a Transfection: methods |2 MeSH |
| 700 | 1 | _ | |a Gschwendtberger, Thomas |b 1 |
| 700 | 1 | _ | |a Felski, Christian |b 2 |
| 700 | 1 | _ | |a Jahn, Christopher |0 0000-0002-0226-2464 |b 3 |
| 700 | 1 | _ | |a Langer, Florian |b 4 |
| 700 | 1 | _ | |a Sterneckert, Jared |b 5 |
| 700 | 1 | _ | |a Hermann, Andreas |0 P:(DE-2719)2811732 |b 6 |u dzne |
| 700 | 1 | _ | |a Lühmann, Jonathan |0 0000-0002-9446-2411 |b 7 |
| 700 | 1 | _ | |a Steinemann, Doris |0 0000-0001-8797-0816 |b 8 |
| 700 | 1 | _ | |a Haase, Alexandra |b 9 |
| 700 | 1 | _ | |a Martin, Ulrich |0 0000-0003-1058-4540 |b 10 |
| 700 | 1 | _ | |a Petri, Susanne |b 11 |
| 700 | 1 | _ | |a Cantz, Tobias |0 0000-0002-1382-9577 |b 12 |
| 773 | _ | _ | |a 10.1038/s41598-021-01689-2 |g Vol. 11, no. 1, p. 22154 |0 PERI:(DE-600)2615211-3 |n 1 |p 22154 |t Scientific reports |v 11 |y 2021 |x 2045-2322 |
| 856 | 4 | _ | |y OpenAccess |u https://pub.dzne.de/record/163639/files/DZNE-2022-00385.pdf |
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