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000163639 1001_ $$00000-0003-3588-7847$$aEggenschwiler, Reto$$b0
000163639 245__ $$aA selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.
000163639 260__ $$a[London]$$bMacmillan Publishers Limited, part of Springer Nature$$c2021
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000163639 520__ $$aCRISPR 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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000163639 650_7 $$0EC 1.15.1.1$$2NLM Chemicals$$aSuperoxide Dismutase-1
000163639 650_2 $$2MeSH$$aAmyotrophic Lateral Sclerosis: genetics
000163639 650_2 $$2MeSH$$aCRISPR-Cas Systems
000163639 650_2 $$2MeSH$$aCell Line
000163639 650_2 $$2MeSH$$aGene Editing: methods
000163639 650_2 $$2MeSH$$aHEK293 Cells
000163639 650_2 $$2MeSH$$aHumans
000163639 650_2 $$2MeSH$$aInduced Pluripotent Stem Cells: metabolism
000163639 650_2 $$2MeSH$$aMutation
000163639 650_2 $$2MeSH$$aSuperoxide Dismutase-1: genetics
000163639 650_2 $$2MeSH$$aTransfection: methods
000163639 7001_ $$aGschwendtberger, Thomas$$b1
000163639 7001_ $$aFelski, Christian$$b2
000163639 7001_ $$00000-0002-0226-2464$$aJahn, Christopher$$b3
000163639 7001_ $$aLanger, Florian$$b4
000163639 7001_ $$aSterneckert, Jared$$b5
000163639 7001_ $$0P:(DE-2719)2811732$$aHermann, Andreas$$b6$$udzne
000163639 7001_ $$00000-0002-9446-2411$$aLühmann, Jonathan$$b7
000163639 7001_ $$00000-0001-8797-0816$$aSteinemann, Doris$$b8
000163639 7001_ $$aHaase, Alexandra$$b9
000163639 7001_ $$00000-0003-1058-4540$$aMartin, Ulrich$$b10
000163639 7001_ $$aPetri, Susanne$$b11
000163639 7001_ $$00000-0002-1382-9577$$aCantz, Tobias$$b12
000163639 773__ $$0PERI:(DE-600)2615211-3$$a10.1038/s41598-021-01689-2$$gVol. 11, no. 1, p. 22154$$n1$$p22154$$tScientific reports$$v11$$x2045-2322$$y2021
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