OR03
PB-PE, a piggyBac prime editing platform for efficient evaluation of optimal CRISPR-PE designs
R Eggenschwiler¹ T Gschwendtberger¹ C Jahn¹ S McCallan¹ J Hoepfner¹ C Baer¹ S Petri¹ T Cantz¹
1:Hannover Medical School
CRISPR prime editors (PE) are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering as well as a number of advantages over DNA base-editors. However, efficiency and accuracy of PE is largely dependent on parameters such as targeted PAM site, exact PE enzyme and pegRNA design. Evaluation of these parameters in a given target cell line of choice generally requires highly efficient delivery of vectors and good transfection protocols are often unavailable. To this end, we introduce piggyBac prime editors (PB-PE), which allow for selection-based sustained expression of ‘all-in-one’ PE. We apply PB-PE for efficient editing of notoriously difficult-to-transfect hiPSC, improving fraction of edited cells by up to 10-fold when compared to transient techniques and achieving more than 50% overall efficiency in a bulk population. We demonstrate proof-of-concept for the PB-PE system in a synthetic traffic light reporter gene, as well as at disease-related genomic loci in the human SOD1 and GLA genes. A variety of PB-PE vectors can target diverse PAM motifs such as NG, NGG, NGNG, NGTC, NR/YN and NAAN. Additionally, the PB-PE method can be employed to give a quick report about activity and accuracy of chosen pegRNA designs after just a few days of expression. Finally, PB-PE vectors can be removed with an integrase-deficient PB transposase and excised cells can be enriched by fialuridine selection. Together, we present a versatile PE toolbox, which can be robustly used in hard-to-transfect cell lines.
