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.
Highlights
Addition of CRISPR prime editors (PEs) to the repertoire of tools for directed genome editing has opened new avenues in life sciences and biomedical applications
We find that piggyBac prime-editing (PB-PE) corrects more than 50% of cells with efficient prime editing gRNAs (pegRNAs), even when applying non-optimized transfection protocols, in both, HEK293 and hiPSC
Proper functionality of a traffic light reporter (TLR) bulk population requires that a large fraction of the cells can be activated and that potential dual activation resulting from cells with multiple reporter integrations is at very low level
Summary
Addition of CRISPR prime editors (PEs) to the repertoire of tools for directed genome editing has opened new avenues in life sciences and biomedical applications. Previous CRISPR/Cas[9] approaches that were based on the repair of Cas9-induced DNA double strand breaks via non-homologous end joining (NHEJ) or homology drivenrepair (HDR) exhibit limitations with respect to undesired off-target DNA-cleavage and overall DNA editing efficacies Another approach is utilizing engineered Cas-proteins that allow for direct DNA editing as base editors (BEs) by mediating highly efficient transition of purine (A ↔ G) or pyrimidine (C ↔ T) bases with low rates of unintended indel formation. We implemented PB-PE in a practical-oriented manner for fast and efficient editing of an R115G point mutation in the human superoxide dismutase 1 (SOD1) gene of patient-derived hiPSC This mutation is associated with certain cases of amyotrophic lateral sclerosis (ALS)[4]. We present PB-PE as an exciting tool for fast and simple prediction of suitable pegRNA designs as well as for more convenient application of prime editors as it is less dependent on optimization of transfection for each cell line
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
