| Autor: |
Kondrashov A; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Duc Hoang M; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Smith JGW; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Bhagwan JR; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Duncan G; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Mosqueira D; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Munoz MB; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Vo NTN; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom ., Denning C; Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom . |
| Abstrakt: |
Modeling disease with human pluripotent stem cells (hPSCs) is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While "footprint-free" Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. In this study, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across four cardiac-associated loci (ADRB2, GRK5, RYR2, and ACTC1) by genomic cleavage and editing efficiencies of 8%-93% and 8%-67%, respectively, including mono- and/or biallelic events. Pluripotency was retained, as was differentiation into high-purity cardiomyocytes (CMs; 88%-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the β-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the β-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies. |
