Introduction: Genome-wide sequencing has uncovered numerous novel mutations in candidate genes associated with congenital heart disease (CHD). However, identifying the causal factors among statistically associated genes requires their experimental validation.

Materials and results: To determine the functional role of novel human CHD candidate genes, we performed a CRISPR-Cas9-based knockout screen using the small fish model system medaka (Oryzias latipes). We investigated a selection of 12 recently published genes from sequencing studies in CHD patients that lacked in-vivo experimental evidence to demonstrate the relevance for cardiac development. Single guide RNAs (sgRNAs) targeting the conserved medaka orthologs were co-injected with Cas9 mRNA into 1-cell stage fertilized eggs to induce targeted knockout. Injected embryos (crispants) were screened for cardiovascular defects at 4 and 6 days post-fertilization using standardized criteria. Targeting of myrf, a transcription factor primarily linked to myelination, resulted in the most striking phenotypes. Medaka myrf crispants showed a high degree (27%) of specific cardiovascular defects (impaired cardiac looping, a characteristic inflection of the heart, and a spectrum of underdeveloped cardiac muscle; n = 106) not found in controls. Confocal microscopy demonstrated these cardiac defects at a cellular scale. To validate our findings, 2 sets of sgRNAs targeting the positions of published patient mutations in MYRF recapitulated the specific phenotype from the screen (51% and 43% myrf-knockout phenotypes for sgRNAs set 1 (n=49) and set 2 (n=44) versus 0% phenotypes in control (n=48 and n=48, respectively)). In a stable myrf knockout line, with a 182 bp deletion in myrf's C-terminal region, homozygous offspring recapitulated the specific cardiac defects. We targeted a conserved amino acid corresponding to Phe387 in humans using adenine base editing, shown to be mutated in a patient and contained in the DNA binding domain of MYRF. Base editing the orthologous Phe387 locus induced a significant rate of looping defects and cardiac edema compared to control-injected embryos.

Conclusion: This study indicates that the transcription factor MYRF has an essential role in cardiac development and that missense mutations in MYRF are sufficient to cause cardiovascular defects. With the created myrf knockout medaka line, we provide a novel animal model for a detailed mechanistic analysis of how MYRF functions control cardiac development.