Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells
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 simplifie...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Mary Ann Liebert
2018
|
| Subjects: | |
| Online Access: | https://eprints.nottingham.ac.uk/49543/ |
| _version_ | 1848798020502552576 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Kondrashov, Alexander |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:13:08Z |
| format | Article |
| id | nottingham-49543 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:13:08Z |
| publishDate | 2018 |
| publisher | Mary Ann Liebert |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-495432019-03-15T04:30:18Z https://eprints.nottingham.ac.uk/49543/ 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 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. Mary Ann Liebert 2018-03-15 Article PeerReviewed application/pdf en https://eprints.nottingham.ac.uk/49543/1/REVISED%2C%20COLLATED%20%26%20SUBMITTED_Stem%20Cells%20%26%20Development%20%285-Feb-2018%29.pdf 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 Cas9/CRISPR; PiggyBac; gene editing; human pluripotent stem cells; genetic disease modelling; cardiomyocytes http://online.liebertpub.com/doi/abs/10.1089/scd.2017.0268 doi:10.1089/scd.2017.0268 doi:10.1089/scd.2017.0268 |
| spellingShingle | Cas9/CRISPR; PiggyBac; gene editing; human pluripotent stem cells; genetic disease modelling; cardiomyocytes 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 Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title | Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title_full | Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title_fullStr | Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title_full_unstemmed | Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title_short | Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells |
| title_sort | simplified footprint-free cas9/crispr editing of cardiac-associated genes in human pluripotent stem cells |
| topic | Cas9/CRISPR; PiggyBac; gene editing; human pluripotent stem cells; genetic disease modelling; cardiomyocytes |
| url | https://eprints.nottingham.ac.uk/49543/ https://eprints.nottingham.ac.uk/49543/ https://eprints.nottingham.ac.uk/49543/ |