GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR- Cas9 -mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

Non-alcoholic fatty liver disease is a huge cause of chronic liver failure around the world. This condition has become more prevalent as rates of metabolic syndrome, type 2 diabetes, and obesity have also escalated. The unfortunate outcome for many people is liver cirrhosis that warrants transplantation or being unable to receive a transplant since many livers are discarded due to high levels of steatosis. Over the past several years, however, a great deal of work has gone into understanding the pathophysiology of this disease as well as possible treatment options. This review summarizes various defatting strategies including in vitro use of pharmacologic agents, machine perfusion of extracted livers, and genomic approaches targeting specific proteins. The goal of the field is to reduce the number of necessary transplants and expand the pool of organs available for use.

32GABA type-A (GABA-A) receptors containing the α2 subunit (Gabra2) are expressed in most brain regions and are 33 critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, 34 affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is 35 modulated by sequence variants in several brain structures and populations, including F2 crosses originating from 36 C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global 37 reduction of Gabra2 brain mRNA and protein in the B6J strain relative to other inbred strains, and identify and validate 38 the causal mutation in B6J. The mutation is a single base pair intronic deletion located adjacent to a splice acceptor site 39 that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now 40 pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of 41 consomic lines. Repair of the deletion using CRISPR-Cas9 mediated gene editing on a B6J genetic background completely 42 restored brain levels of Gabra2 mRNA and protein. Comparison of transcript expression in hippocampus, cortex, and 43 striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in 44 striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or 45 inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal 46 function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic 47 studies in neurobiological research as this strain is widely used to generate genetically engineered mice and murine genetic 48 populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug 49

No abstract

CRISPR gene editing is a molecular technology that can be used to silence gene expression. In this experiment, genes that are known to play a role in lipid accumulation in hepatocytes were targeted. Specifically, levels of fatty acid transport proteins 2 and 5 (FATP2 & 5) have been shown to be elevated in cases of non-alcoholic fatty liver disease. The goal of this experiment was to reduce expression of these genes by using a dead Cas9 (dCas9) protein with an attached inhibitory domain (KRAB) that acts on the promotor region. When measuring the mRNA expression, it was determined that the levels of the CRISPR-modified gene products were significantly reduced compared to the control. However, the same extent of inhibition was not consistently observed when conducting flow cytometry. Current work is aimed at discovering why lipid accumulation is not inhibited to the expected degree based on the results of mRNA expression.