• Slide 1
  • RyRs

    Traces illustrating RYR single channel activity in the planar lipid bilayer recorded on the Orbit 16.

2018 - Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells

Icon CE  CardioExcyte 96 publication in Stem Cells and Development

Authors:
Kondrashov A., Hoang M.D., Smith J., Bhagwan J., Duncan G., Mosqueira D., Munoz M., Vo N.T.N., Denning C.

Journal:
Stem Cells and Development (2018) ahead or print, doi.org/10.1089/scd.2017.0268


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 b-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the b-blocker, propranolol. This simplified, footprint-free approach will be useful for mechanistic studies.


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