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Kondrashov, Alexander, Hoang, Minh Duc, Smith, James G. W. 
ORCID: https://orcid.org/0000-0003-0427-8678, Bhagwan, Jamie R., Duncan, Gary, Mosqueira, Diogo, Munoz, Maria Barbadillo, Vo, Nguyen T. N. and Denning, Chris
(2018)
Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells.
Stem Cells and Development, 27 (6).
pp. 391-404.
ISSN 1557-8534
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Abstract
Modelling disease with 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. Here, 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 4 cardiac-associated loci (ADRB2, GRK5, RYR2, ACTC1) by genomic cleavage and editing efficiencies of 8-93% and 8-67 respectively, including mono- and/or bi-allelic 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 beta-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the beta-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | crispr,piggybac,gene editing,human pluripotent stem cells,genetic disease modelling,cardiomyocytes,sdg 3 - good health and well-being ,/dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Groups > Cardiovascular and Metabolic Health Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 09 Jan 2019 10:30 |
| Last Modified: | 19 Oct 2023 02:20 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/69496 |
| DOI: | 10.1089/scd.2017.0268 |
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