Simplified Footprint-Free Cas9/CRISPR Editing of Cardiac-Associated Genes in Human Pluripotent Stem Cells

Abstract

Modeling disease with human pluripotent stem cells (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. In this study, 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 four cardiac-associated loci (ADRB2, GRK5, RYR2, and ACTC1) by genomic cleavage and editing efficiencies of 8%–93% and 8%–67%, respectively, including mono- and/or biallelic 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 β-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the β-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies.

Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy

“…To address the highly complex genetic causation associated with HCM, isogenic hPSC-CM lines were previously generated by CRISPR/Cas9 genome editing technology [ 32 ], which isolates the impact of the mutation of interest. This approach was used to introduce the p.R453C-β-MHC mutation [ 19 ] in three healthy (WT/WT) hPSC lines (AT1-hiPSC, REBL-PAT-hiPSC and HUES7-hESC), resulting in heterozygote (MUT/WT) and homozygote (MUT/MUT) variants, and a β-MHC-knockout (KO/KO) ( Figure 1 a) in a total of 10 cell lines.…”

Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits

“…Isogenic controls were generated for E99K1, NC, and E99K2 hiPSCs using a footprint-free PiggyBac based CRISPR/Cas9strategy, coupled with PCR screening and sequence confirmation ( Kondrashov et al., 2018 ) ( Figure 2 A). This identified targeted clones for E99K1 and E99K2 where the c.301G>A ACTC1 mutation had been corrected (producing E99K1-Corr and E99K2-Corr) and introduced into one allele of NC (producing NC-Edit-E99K) ( Figure 2 B).…”

“…The strategy for correcting/introducing the E99K-ACTC1 mutation to generate isogenic control lines has been previously described in detail ( Kondrashov et al., 2018 ). In brief, an ACTC1 targeting vector was constructed containing a dual drug selection cassette (Puro-ΔTK) flanked by PiggyBac recombination sites, and the two ACTC1 sequences (1 kb upstream and 1 kb downstream) homologous to the endogenous target locus cut site.…”

Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

“…The other comprised a pair that were heterozygous originally patient-derived ( ACTC1 WT/MUT ) and corrected ( ACTC1 WT/WT ) for the c. ACTC1 G301A mutation 21 . for the c. ACTC1 G301A mutation and CRISPR Cas9 corrected ( ACTC1 WT/WT ) 21 , 28 .…”