CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease

Abstract

High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.

Hypoplastic Left Heart Syndrome: Signaling & Molecular Perspectives, and the Road Ahead

“…According to the published data, the individuals diagnosed with HLHS exhibit de novo mutations in both the N-terminal and C-terminal segments of the MYRF protein, indicating a complex underlying mechanism. The association between Myrf and HLHS has been further investigated using CRISPR-Cas9 technology in the vertebrate medaka model, and the Myrf mutant line exhibited a significantly prominent hypoplastic ventricle, which closely recapitulates the phenotypes observed in pediatric patients [129]. Overall, the signaling mechanism by which Myrf contributes to the pathogenesis of HLHS remains unclear, and there is limited knowledge in this area.…”

“…Multiple studies reported that point mutations of myelin regulatory factor (MYRF) are associated with the occurrence of HLHS [129][130][131], suggesting the possibility that monogenic mutations can cause HLHS. Myrf is a vital membrane-bound transcription factor involved in the development of the urogenital, neural, visual, and cardiac systems [132][133][134][135][136].…”