Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells

Kondrashov, Alexander, Hoang, Minh Duc, Smith, James G. W. ORCID:, 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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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
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Depositing User: LivePure Connector
Date Deposited: 09 Jan 2019 10:30
Last Modified: 21 Oct 2022 21:33
DOI: 10.1089/scd.2017.0268

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