Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening and without introducing exogenous sequence at the locus of interest or marker mutations elsewhere. To this end, we describe a coconversion strategy, using CRISPR/Cas9 in which screening for a dominant phenotypic oligonucleotide-templated conversion event at one locus can be used to enrich for custom modifications at another unlinked locus. After the desired mutation is identified among the F 1 progeny heterozygous for the dominant marker mutation, F 2 animals that have lost the marker mutation are picked to obtain the desired mutation in an unmarked genetic background. We have developed such a coconversion strategy for Caenorhabditis elegans, using a number of dominant phenotypic markers. Examining the coconversion at a second (unselected) locus of interest in the marked F 1 animals, we observed that 14-84% of screened animals showed homologous recombination. By reconstituting the unmarked background through segregation of the dominant marker mutation at each step, we show that custom modification events can be carried out recursively, enabling multiple mutant animals to be made. While our initial choice of a coconversion marker [rol-6(su1006)] was readily applicable in a single round of coconversion, the genetic properties of this locus were not optimal in that CRISPR-mediated deletion mutations at the unselected rol-6 locus can render a fraction of coconverted strains recalcitrant to further rounds of similar mutagenesis. An optimal marker in this sense would provide phenotypic distinctions between the desired mutant/+ class and alternative +/+, mutant/null, null/null, and null/+ genotypes. Reviewing dominant alleles from classical C. elegans genetics, we identified one mutation in dpy-10 and one mutation in sqt-1 that meet these criteria and demonstrate that these too can be used as effective conversion markers. Coconversion was observed using a variety of donor molecules at the second (unselected) locus, including oligonucleotides, PCR products, and plasmids. We note that the coconversion approach described here could be applied in any of the variety of systems where suitable coconversion markers can be identified from previous intensive genetic analyses of gain-of-function alleles.T YPE II CRISPR/Cas9 bacterial immunity systems provide programmable DNA endonuclease activities that have recently revolutionized genome editing in a wide range of organisms (Wang et al. 1999; Chiu et al. 2013;Cho et al. 2013;Dicarlo et al. 2013;Friedland et al. 2013;Gratz et al. 2013;Jiang et al. 2013;Katic and Großhans 2013;Li et al. 2013;Lo et al. 2013;Nekrasov et al. 2013;Kim et al. 2014;Zhao et al. 2014). Recognition by the Cas9 protein entails two sequence elements in the target: a protospacer adjacent motif (PAM) (NGG for Streptococcus pyogenes Cas9) and a re...

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SummaryThe etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle‐aged Tet‐mev‐1 mice with the SDHCV69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle‐aged Tet‐mev‐1 mice overproduced MitoSOX Red‐detectable mitochondrial ROS compared to age‐matched wild‐type C57BL/6J mice, only young adult Tet‐mev‐1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn‐ and Cu/Zn‐SODs) activities to eliminate the MitoSOX Red‐detectable mitochondrial ROS. In contrast, middle‐aged Tet‐mev‐1 mice accumulated both MitoSOX Red‐detectable mitochondrial ROS and CM‐H2DCFDA‐detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle‐aged Tet‐mev‐1 mice relative to age‐matched wild‐type C57BL/6J mice. Furthermore, only middle‐aged Tet‐mev‐1 mice showed JNK/SAPK activation and Ca2+ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100β in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age‐dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.

Mitochondria play a role of energy production and produce intracellular reactive oxygen species (ROS), especially superoxide anion (O2(-)) as a byproduct of energy metabolism at the same time. O2(-) is converted from oxygen and is overproduced by excessive electron leakage from the mitochondrial respiratory chain. It is well known that mitochondrial complexes I and III in the electron transport system are the major endogenous ROS sources. We have previously demonstrated that mutations in complex II can result in excessive ROS (specifically in SDHC: G71E in Caenorhabditis elegans, I71E in Drosophila and V69E in mouse). Moreover, this results in premature death in C. elegans and Drosophila as well as tumorigenesis in mouse embryonic fibroblast cells. In humans, it has been reported that mutations in SDHB, SDHC or SDHD, which are the subunits of mitochondrial complex II, often result in inherited head and neck paragangliomas (PGLs). Recently, we established Tet-mev-1 conditional transgenic mice using our uniquely developed Tet-On/Off system, which can induce the mutated SDHC gene to be equally and competitively expressed compared to the endogenous wild-type SDHC gene. These mice experienced mitochondrial respiratory chain dysfunction that resulted in oxidative stress. The mitochondrial oxidative stress caused excessive apoptosis in several tissues leading to low-birth-weight infants and growth retardation during neonatal developmental phase in Tet-mev-1 mice. Tet-mev-1 mice also displayed precocious age-dependent corneal physiological changes, delayed corneal epithelialization, decreased corneal endothelial cells, thickened Descemet's membrane and thinning of parenchyma with corneal pathological dysfunctions such as keratitis, Fuchs' corneal dystrophy (FCD) and probably keratoconus after the normal development and growth phase. Here, we review the relationships between mitochondrial oxidative stress and phenomena in mev-1 animal models with mitochondrial complex II SDHC mutations. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

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