Kondrashov, Alexander, Hoang, Minh Duc, Smith, James G.W., Bhagwan, Jamie R., Duncan, Gary, Mosqueira, Diogo, Barbadillo Munoz, Maria, 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
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: |
15 Mar 2019 04:30 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/49543 |
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