CRISPR-Cas9 mediated knockout of NDUFS4 in human iPSCs: A model for mitochondrial complex I deficiency

Abstract

Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4-related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD⁺ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD⁺ modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo, these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction.

Keywords: CI deficiency; CRISPR-Cas9; Mitochondrial disease; iPSC.