Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several nonreplicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, we used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, we provide evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

1Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct 2 developmental defects, including primordial dwarfism, small ears, and small or missing 3 patella. Genes encoding members of the origin recognition complex (ORC) and additional 4 proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, and MCM5) are 5 mutated in individuals diagnosed with MGS. The primary role of ORC is to license origins 6 during the G1 phase of the cell cycle, but it also plays roles in cilia development, 7 heterochromatin formation, and other cellular processes. Because of its essential role in 8 DNA replication, ORC is required for every cell division during development. Thus, it is 9 unclear how the Meier-Gorlin syndrome mutations in ORC lead to the tissue-specific 10 defects associated with the disease. To address this question, we have used Cas9-11 mediated genome engineering to generate a Drosophila melanogaster model of 12 individuals carrying a mutation in ORC4. Like the people with Meier-Gorlin syndrome, 13 these flies reach adulthood, but have several tissue-specific defects. Genetic analysis 14revealed that this allele is a hypomorph and that mutant females are sterile. We 15 demonstrated that this sterility is caused by a failure in DNA replication. By leveraging the 16 well-studied Drosophila system, we showed that a disease-causing mutation in orc4 17 disrupts DNA replication, and we propose that in individuals with MGS defects arise 18 preferentially in tissues with a high-replication demand. 19 20 4