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

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Authors

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

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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.

Details

Original languageEnglish
Pages (from-to)391-404
Number of pages14
JournalStem Cells and Development
Volume27
Issue number6
Early online date12 Mar 2018
DOIs
Publication statusPublished - 12 Mar 2018
Peer-reviewedYes

Keywords

    Research areas

  • Cas9/CRISPR, PiggyBac, gene editing, human pluripotent stem cells, genetic disease modelling, cardiomyocytes

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