History of genome editing: From meganucleases to CRISPR.

CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this groundbreaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease

Successful use of HTF as a basal fertilization medium during SEcuRe mouse in vitro fertilization

Abstract Objective The ever-increasing number of genetically engineered mouse models highlights the need for efficient archiving and distribution of these lines. Sperm cryopreservation has become the preferred technique for the majority of these models due to its low requirement of costs, time, and experimental animals. Yet, current in vitro fertilization (IVF) protocols either exhibit decreased fertilization efficiency for the most popular C57BL/6 strain, as recently demonstrated by us, or require costly and difficult-to-prepare media, respectively. As a result, we previously developed SEcuRe, a modified IVF protocol with low costs and high fertilization efficiency. The popular basal fertilization medium, Cook’s® proprietary “Research vitro fert” (RVF), used in this protocol has recently been discontinued. As a result, the application of the SEcuRe approach and other IVF protocols employing this medium has been severely limited. Results Here we show that human tubal fluid (HTF), a popular and widely available medium with a known composition, can be used as a basal fertilization medium instead of RVF. Comparison of RVF and HTF during 58 independent SEcuRe IVFs with cryopreserved C57BL/6 sperm revealed equal fertilization and live birth rates. In addition, we demonstrate that HTF has a substantially extended shelf-life by utilizing commercial HTF that was six months past its expiration date, yet did not affect fertilization during IVF or subsequent embryo development. This finding not only increases the economic value of our modified method, but also validates it once more. Our results demonstrate that common, shelf-life extended HTF can be used in SEcuRe IVF in place of now-discontinued RVF medium and ensure the applicability of the method, which we since termed SEcuRe 2.0. Our modified SEcuRe 2.0 strategy will assist researchers to efficiently archive and distribute genetically engineered mouse models in a cost-effective, easily adaptable, and 3R-compliant manner with minimal animal use

Live birth rate of offspring upon transgenesis by PNI or EEZy.

<p>Live birth rate of offspring upon transgenesis by PNI or EEZy.</p

Embryo toxicity of EEZy as compared to pronuclear injection.

<p>(A) Assessment of embryo development as percentage of developed blastocysts from zygotes after EEZy. Untreated zygotes are compared to zygotes electroporated with Opti-MEM (Mock) or CRISPR/Cas9 components targeting the <i>Gt(ROSA)26Sor</i> locus (CRISPR). Data represent three independent experiments. (B) Quantification of viable <i>Nphs2</i>-targeted zygotes from four experiments immediately after PNI compared to electroporated zygotes and (C) the correspondingly developed blastocysts. (D) Developed blastocyst calculated from the number of zygotes before transgenesis in (B). (E) Quantification of RFLP genotyping from four independent experiments for HDR efficiency in blastocysts from zygotes after PNI compared to electroporated zygotes from the experiments depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196891#pone.0196891.g001" target="_blank">Fig 1B and 1D</a>, right columns. Data are means ± standard deviation. *p < 0.05, **p < 0.01, ns = non-significant. N = total number of embryos analyzed.</p