Royal College of Surgeons in Ireland
Using Ribonucleoprotein-based CRISPR:Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A ±).pdf (1.98 MB)

Using ribonucleoprotein-based CRISPR?Cas9 to edit single nucleotide on human induced pluripotent stem cells to model type 3 long QT syndrome (SCN5A ±)

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posted on 2023-09-12, 09:58 authored by Ning Ge, Min Liu, Rui Li, Nicholas M Allen, Joseph Galvin, Sanbing Shen, Timothy O'Brien, Terence W Prendiville

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.


Open Access funding provided by the IReL Consortium

National Children’s Research Centre (NCRC)

Galway University Foundation

NUIG College Scholarship

China Scholarship Council



The original article is available at

Published Citation

Ge N, et al. Using ribonucleoprotein-based CRISPR/Cas9 to edit single nucleotide on human induced pluripotent stem cells to model type 3 long QT syndrome (SCN5A ±). Stem Cell Rev Rep. 2023

Publication Date

31 August 2023

PubMed ID



  • FutureNeuro Centre


Springer Nature


  • Published Version (Version of Record)