NRF2 activation inhibits valproic acid-induced neural tube defects in mice.

Valproic acid (VPA) is a widely prescribed medication that has traditionally been used to treat epilepsy, yet embryonic exposure to VPA increases the risk of the fetus developing neural tube defects (NTDs). While the mechanism by which VPA causes NTDs is unknown, we hypothesize that VPA causes dysmorphogenesis through the disruption of redox-sensitive signaling pathways that are critical for proper embryonic development, and that protection from the redox disruption may decrease the prevalence of NTDs. Time-bred CD-1 mice were treated with 3H-1,2-dithiole-3-thione (D3T), an inducer of nuclear factor erythroid 2-related factor 2 (NRF2)-a transcription factor that activates the intracellular antioxidant response to prevent redox disruptions. Embryos were then collected for whole embryo culture and subsequently treated with VPA in vitro. The glutathione (GSH)/glutathione disulfide (GSSG) redox potential (Eh), a measure of the intracellular redox environment, was measured in the developing mouse embryos. Embryos treated with VPA exhibited a transiently oxidizing GSH/GSSG Eh, while those that received D3T pretreatment prior to VPA exposure showed no differences compared to controls. Moving to an in utero mouse model, time-bred C57BL/6 J dams were pretreated with or without D3T and then exposed to VPA, after which all embryos were collected for morphological analyses. The prevalence of open neural tubes in embryos treated with VPA significantly decreased with D3T pretreatment, as did the severity of the observed defects evaluated by a morphological assessment. These data show that NRF2 induction via D3T pretreatment protects against VPA-induced redox dysregulation and decreases the prevalence of NTDs in developing mouse embryos.

Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools. We compared plasmid, ribonucleoprotein (RNP), and RNA transfection to determine which was method was most optimal for editing cells in a laboratory setting. We limited our comparison to lipofection-mediated introduction because the reagents are widely available. To facilitate optimization, we developed a novel reporter assay to measure gene disruption and the introduction of a variety of exogenous DNA tags. Each method efficiently disrupted endogenous genes and was able to stimulate the introduction of foreign DNA at specific sites, albeit to varying efficiencies. RNP transfection produced the highest level of gene disruption and was the most rapid and efficient method overall. Finally, we show that very short homology arms of 30 base pairs can mediate site-specific editing. The methods described here should broaden the accessibility of RNP-mediated lipofection for laboratory genome-editing experiments.