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

Kondrashov, Alexander; Hoang, Minh Duc; Smith, James G.W.; Bhagwan, Jamie R.; Duncan, Gary; Mosqueira, Diogo; Barbadillo Munoz, Maria; Vo, Nguyen T.N.; Denning, Chris

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.

Citation

Kondrashov, A., Hoang, M. D., Smith, J. G., Bhagwan, J. R., Duncan, G., Mosqueira, D., …Denning, C. (in press). Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells. Stem Cells and Development, 27(6), https://doi.org/10.1089/scd.2017.0268

• Journal Article Type: Article
• Acceptance Date: Feb 5, 2018
• Online Publication Date: Mar 15, 2018
• Deposit Date: Feb 5, 2018
• Publicly Available Date: Mar 16, 2019
• Journal: Stem Cells and Development
• Print ISSN: 1547-3287
• Electronic ISSN: 1557-8534
• Publisher: Mary Ann Liebert
• Peer Reviewed: Peer Reviewed
• Volume: 27
• Issue: 6
• DOI: https://doi.org/10.1089/scd.2017.0268
• Keywords: Cas9/CRISPR; PiggyBac; gene editing; human pluripotent stem cells; genetic disease modelling; cardiomyocytes
• Public URL: https://nottingham-repository.worktribe.com/output/920373
• Publisher URL: http://online.liebertpub.com/doi/abs/10.1089/scd.2017.0268
• Contract Date: Feb 5, 2018