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

Kondrashov, Alexander and Hoang, Minh Duc and Smith, James G.W. and Bhagwan, Jamie R. and Duncan, Gary and Mosqueira, Diogo and Barbadillo Munoz, Maria and 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). 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
Keywords: Cas9/CRISPR; PiggyBac; gene editing; human pluripotent stem cells; genetic disease modelling; cardiomyocytes
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Medicine > Division of Cancer and Stem Cells
Identification Number: 10.1089/scd.2017.0268
Depositing User: Denning, Chris
Date Deposited: 05 Feb 2018 15:29
Last Modified: 26 Mar 2018 13:35
URI: http://eprints.nottingham.ac.uk/id/eprint/49543

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