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: 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

[thumbnail of SCD_Kondrashov_etal_2018]
Preview
PDF (SCD_Kondrashov_etal_2018) - Published Version
Available under License Creative Commons Attribution.

Download (691kB) | Preview

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

Downloads

Downloads per month over past year

Actions (login required)

View Item View Item