The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Estrogen Receptor ß as a Candidate Regulator of Sex Differences in the Maternal Immune Activation Model of ASD.

Interestingly, more males are diagnosed with autism spectrum disorder (ASD) than females, yet the mechanism behind this difference is unclear. Genes on the sex chromosomes and differential regulation by sex steroid hormones and their receptors are both candidate mechanisms to explain this sex-dependent phenotype. Nuclear receptors (NRs) are a large family of transcription factors, including sex hormone receptors, that mediate ligand-dependent transcription and may play key roles in sex-specific regulation of immunity and brain development. Infection during pregnancy is known to increase the probability of developing ASD in humans, and a mouse model of maternal immune activation (MIA), which is induced by injecting innate immune stimulants into pregnant wild-type mice, is commonly used to study ASD. Since this model successfully recaptures the behavioral phenotypes and male bias observed in ASD, we will discuss the potential role of sex steroid hormones and their receptors, especially focusing on estrogen receptor (ER)ß, in MIA and how this signaling may modulate transcription and subsequent inflammation in myeloid-lineage cells to contribute to the etiology of this neurodevelopmental disorder.

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.