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

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 pegRN

SUBMITTER: Eggenschwiler R 

PROVIDER: S-EPMC8589839 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature