Adenine base editing of the RBM20 p.P638L mutation in hiPSC-derived cardiomyocytes decreased missplicing and enhanced nuclear relocalization of RBM20, despite minor bystander editing.
Does adenine base editing improve splicing and nuclear relocalization of RBM20 in hiPSC-derived cardiomyocytes with the RBM20 p.P638L mutation?
Adenine base editing successfully corrects the RBM20 p.P638L mutation in hiPSC-derived cardiomyocytes, rescuing splicing and protein localization defects, though precision remains a challenge due to bystander editing.
Abstract Background/Purpose The RNA binding motif protein 20 (RBM20), acts as a cardiac splicing factor. The majority of the known cardiomyopathy-associated RBM20 mutations are localized in the highly conserved RS domain. These mutations lead to a mislocalization of the protein from the nucleus to the cytoplasm, followed by mis-splicing of targets like TTN or RYR2. We aimed to establish and apply base editing to correct the pathogenic RBM20 p.P638L mutation within the RS domain, as base editing is a potent gene editing tool even in postmitotic cells like cardiomyocytes. Methods Using CRISPR/Cas9 we generated human induced pluripotent stem cell (hiPSC) lines which are either heterozygous or homozygous for the pathogenic RBM20 p.P638L mutation. For mutation correction, adenine base editing was performed. We evaluated two adenine base editors (ABE) in hiPSCs: a conventional ABE (cABE) and a nearly PAM-less ABE (ABEpl). We constructed an all-in-one vector which carries both the base editor and the scaffold for guide RNA expression, to enhance the editing efficiency. Edited hiPSCs were differentiated to hiPSC-derived cardiomyocytes (hiPSC-CMs) and matured. Editing efficiency was further quantified through i) splicing analysis of RBM20 target genes by quantitative real-time PCR and ii) subcellular localization analysis of RBM20. Results Both ABEs were capable to edit the desired mutation. However, cABE usage led to an additional editing event, resulting in the p.V639A variant, which in turn led to partial mislocalization of RBM20 in vitro. Accordingly, ABEpl was examined, enabling a more flexible selection of guide RNA and editing window. Two guides were found to be efficacious in the correction of the p.P638L mutation. However, sequencing also revealed the additional p.V639A editing for one guide (g1). Notably, the other guide (g2) only introduced additional silent variants when evaluated with Sanger sequencing. For precise evaluation we performed amplicon sequencing, which interestingly revealed minor amounts of p.V639A variant (6%) for g2-editing. Nevertheless, edited hiPSC-CMs exhibited decreased missplicing and enhanced nuclear relocalization of RBM20 compared to the untreated RBM20-p.P638L. Conclusion RBM20 mutations are associated with severe forms of DCM, with heart transplantation being the only curative treatment. Therefore, base editing may be a helpful tool in phenotypic rescue. Nevertheless, application and guide RNA selection necessitates meticulous evaluation. For RBM20, the RS-domain mutation hotspot is rich on adenines, which makes a precise editing more difficult. Therefore, the editing evaluation process is important, as only minor amounts of undesired missense editing can be deleterious. Notably, Amplicon sequencing revealed some bystander editing which could not be detected in Sanger sequencing beforehand. However, splicing and localization analysis of edited cells also demonstrated the potential and possibilities of ABEs.
Gross et al. (Fri,) conducted a other in Cardiomyopathy (RBM20 p.P638L mutation). Adenine base editing (cABE and ABEpl) vs. Untreated RBM20-p.P638L hiPSCs was evaluated on Editing efficiency, splicing analysis of RBM20 target genes, and subcellular localization of RBM20. Adenine base editing of the RBM20 p.P638L mutation in hiPSC-derived cardiomyocytes decreased missplicing and enhanced nuclear relocalization of RBM20, despite minor bystander editing.