CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes

Abstract

During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β 2 AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β 2 AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β 2 AR signaling dominated over β 1 AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β 2 AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.

The precise magic of CRISPR

“…This result is due to the selection procedure, which secures the presence of active nuclease in the timeframe of editing. The use of a selection marker, which is present on the Cas9/gRNA expression vector rather than on the template vector, assures ‘clean’ single step editing because it does not require an additional cassette removal step, unlike other gene editing strategies, which use a selection marker within the targeting vector itself [14] However, a potential adverse effect of the approach chosen by Li and co‐authors could be random integration of gRNA/Cas9 expression vector in undesirable genome locations. Several other aspects of the strategy could be optimised in the future, such as the lifetime of the active nuclease within the cell, as a longer presence will increase the probability of off‐target activity.…”

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System

“…The CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas)) system has rapidly become a powerful genome editing tool given its highly precise and efficient targeting method and relatively simple implementation. This molecular genetic system is remarkably powerful for modelling genetic disorders in vitro via the generation of isogenic hPSC clones that differ from the WT hPSCs in only the gene of interest [ 17 , 18 ]. In our previous work, we used CRISPR/Cas9 to generate SH3PXD2b knock out cancer cell lines [ 19 ].…”