CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of ...non-coding regulatory elements. Here, we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-κB led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntington’s disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.
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CRISPR technology has exhibited broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Lim and colleagues demonstrate that mutagenizing cis-acting sequences by CRISPR base editors can enable the perturbation of target gene expression.
The extracellular purinergic agonist uridine diphosphate glucose (UDP-G) activates chemotaxis of human neutrophils (PMN) and the recruitment of PMN at the lung level, via P2Y14 purinergic receptor ...signaling. This effect is similar to the activation of PMN with N-formyl-methionyl-leucyl-phenylalanine (fMLP), a mechanism that also triggers the production of superoxide anion and hydrogen peroxide via the NADPH oxidase system. However, the effects of UDP-G on this system have not been studied. Defects in the intracellular phagocyte respiratory burst (RB) cause recurrent infections, immunodeficiency, and chronic and severe diseases in affected patients, often with sepsis and hypoxia. The extracellular activation of PMN by UDP-G could affect the RB and oxidative stress (OS) in situations of inflammation, infection and/or sepsis. The association of PMNs activation by UDP-G with OS and RB was studied. OS was evaluated by measuring spontaneous chemiluminescence (CL) of PMNs with a scintillation photon counter, and RB by measuring oxygen consumption with an oxygen Clark electrode at 37 °C, in non-stimulated cells and after activation (15 min) with lipopolysaccharides (LPS, 2 µg/mL), phorbol myristate acetate (PMA, 20 ng/mL), or UDP-G (100 μM). The stimulation index (SI) was calculated in order to establish the activation effect of the three agonists. After stimulation with LPS or PMA, the activated PMNs (0.1 × 106 cells/mL) showed an increase in CL (35%, p < 0.05 and 56%, p < 0.01, SI of 1.56 and 2.20, respectively). Contrariwise, the stimulation with UDP-G led to a decreased CL in a dose-dependent manner (60%, 25 μM, p < 0.05; 90%, 50–150 μM, p < 0.001). Nonetheless, despite the lack of oxidative damage, UDP-G triggered RB (SI 1.8) in a dose-dependent manner (38–50%, 100–200 μM, p < 0.0001). UDP-G is able to trigger NADPH oxidase activation in PMNs. Therefore, the prevention of OS and oxidative damage observed upon PMN stimulation with UDP-G indicates an antioxidant property of this molecule which is likely due to the activation of antioxidant defenses. Altogether, LPS and UDP-G have a synergistic effect, suggesting a key role in infection and/or sepsis.
Cas13 nucleases are a class of programmable RNA-targeting CRISPR effector proteins that are capable of silencing target gene expression in mammalian cells. Here, we demonstrate that RfxCas13d, a ...Cas13 ortholog with favorable characteristics to other family members, can be delivered to the mouse spinal cord and brain to silence neurodegeneration-associated genes. Intrathecally delivering an adeno-associated virus vector encoding an RfxCas13d variant programmed to target superoxide dismutase 1 (SOD1), a protein whose mutation can cause amyotrophic lateral sclerosis, reduced SOD1 mRNA and protein in the spinal cord by >50% and improved outcomes in a mouse model of the disorder. We further show that intrastriatally delivering an RfxCas13d variant programmed to target huntingtin (HTT), a protein whose mutation is causative for Huntington’s disease, led to a ~50% reduction in HTT protein in the mouse brain. Our results establish RfxCas13d as a versatile platform for knocking down gene expression in the nervous system.
Copper (Cu) is a bioelement essential for a myriad of enzymatic reactions, which when present in high concentration leads to cytotoxicity. Whereas Cu toxicity is usually assumed to originate from the ...metal's ability to enhance lipid peroxidation, the role of oxidative stress has remained uncertain since no antioxidant therapy has ever been effective. Here we show that Cu overload induces cell death independently of the metal's ability to oxidize the intracellular milieu. In fact, cells neither lose control of their thiol homeostasis until briefly before the onset of cell death, nor trigger a consistent antioxidant response. As expected, glutathione (GSH) protects the cell from Cu-mediated cytotoxicity but, surprisingly, fully independent of its reactive thiol. Moreover, the oxidation state of extracellular Cu is irrelevant as cells accumulate the metal as cuprous ions. We provide evidence that cell death is driven by the interaction of cuprous ions with proteins which impairs protein folding and promotes aggregation. Consequently, cells mostly react to Cu by mounting a heat shock response and trying to restore protein homeostasis. The protective role of GSH is based on the binding of cuprous ions, thus preventing the metal interaction with proteins. Due to the high intracellular content of GSH, it is depleted near the Cu entry site, and hence Cu can interact with proteins and cause aggregation and cytotoxicity immediately below the plasma membrane.
Iron Fe(II) and copper Cu(II) overloads in rat brain are associated with oxidative stress and damage. The purpose of this research is to study whether brain antioxidant enzymes are involved in the ...control of intracellular redox homeostasis in the brain of rats male Sprague–Dawley rats (80–90 g) that received drinking water supplemented with either 1.0 g/L of ferrous chloride (
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= 24) or 0.5 g/L cupric sulfate (
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= 24) for 42 days. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione transferase (GT) activities in brain were determined by spectrophotometric methods and NO production by the content of nitrite concentration in the organ. Chronic treatment with Fe(II) and Cu(II) led to a significant decrease of nitrite content and SOD activity in brain. Activity of NADPH oxidase increased with Cu(II) treatment. Concerning Fe(II), catalase and GT activities increased in brain after 28 and 4 days of treatment, respectively. In the case of Cu(II), catalase activity decreased whereas GT activity increased after 2 and 14 days, respectively. The regulation of redox homeostasis in brain involves changes of the activity of these enzymes to control the steady state of oxidant species related to redox signaling pathways upon Cu and Fe overload. NO may serve to detoxify cells from superoxide anion and hydrogen peroxide with the concomitant formation of peroxynitrite. However, the latest is a powerful oxidant which leads to oxidative modifications of biomolecules. These results suggest a common pathway to oxidative stress and damage in brain for Cu(II) and Fe(II).
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Iron Fe(II) and copper Cu(II) ions produced liver oxidative stress and damage, and as a consequence, changes in the antioxidant protection. The objective of this work is to evaluate whether control ...of redox homeostasis in chronic overload of Fe(II) and Cu(II) is associated with nitric oxide (NO) and antioxidant enzymes protection in liver. Male Sprague–Dawley rats of 80–90 g received the standard diet ad libitum and drinking water supplemented with either 1.0 g/L of ferrous chloride (0.1% w/v,
n
= 24) or 0.5 g/L cupric sulfate (0.05% w/v,
n
= 24) for 42 days. The activities of the enzymes involved in the control of cellular redox homeostasis, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), were determined by spectrophotometric methods, and NO production was determined by the determination of nitrite levels in liver. Chronic overload with Fe(II) and Cu(II) led to a significant increase of NO production while hampering the activity of NADPH oxidase. Meanwhile, the animals supplemented with Fe(II) showed a decrease in SOD and Gpx activities in liver homogenates with respect to baseline activity after 7 days of treatment, whereas the rats which received Cu(II) showed an increased SOD and catalase activity after 28 and 7 days of chronic overload. Further research is required to understand whether the modulation of the activity of these enzymes upon Cu and Fe overload is involved in a common toxic pathway or may serve to control the steady state of oxidant species related to redox signaling pathways.
Graphical abstract
Copper is a highly reactive element involved in a myriad of biological reactions. Thus, while essential for mammalian cells, its concentrations must be kept in check in order to avoid toxicity. This ...metal participates in redox reactions and may exacerbate oxidative stress in aerobic organisms. Nonetheless, the actual driving force of copper-induced cell death is yet unknown. Likely, free copper ions may target different biomolecules that are crucial for the proper functioning of an organism. In this work, we show that free copper induces protein aggregation in serum. The wide set of proteins present in these biological samples are not equally prone to copper-induced aggregation and some, such as albumin, are highly resistant, whereas γ-globulins are highly sensitive. The identity of the proteins in the aggregates becomes fairly homogeneous as metal concentrations go as low as 20 μM. The identification of the proteins by mass spectrometry indicates a preponderance of IgG and a minor presence of other different proteins. Therefore, free copper in blood may contribute to the formation of circulating protein aggregates with a core of IgG. This may impact health not only due to the activity of aggregated IgG but also due to the many proteins co-aggregated. Understanding whether the γ-globulin core and the heterogeneous subgroup of proteins elicit differential responses in the organisms requires further research.
Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic ...effects of Cu2+ and Fe3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu2+ and Fe3+ produced decreases in O2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C50) 60μM Cu2+ and 1.25mM Fe3+), and with succinate as complex II substrate: 64–69% with C50 of 50μM Cu2+ and with C50 of 1.25mM of Fe3+. Respiratory control decreased with Cu2+ (C50 50μM) and Fe3+ (C50 1.25-1-75mM) with both substrates. Cu2+ produced a 2-fold increase and Fe3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25μM Cu2+ (C50 40μM) and from 100μM Fe3+ (C50 1.75mM). Supplementations with Cu2+ and Fe3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death.
The Copper(II) and Iron(III) addition to isolated rat liver mitochondria produced decreased rates of mitochondrial respiration and oxygen consumption in active state 3 and phospholipid peroxidation by a Fenton/Haber Weiss mechanism of oxidative damage. Slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. Display omitted
•Cu(II) and Fe(III) addition to isolated rat liver mitochondria produced inhibition of mitochondrial respiration.•Decreased mitochondrial respiration is maximal in O2 consumption in active state 3.•Mitochondrial respiratory dysfunction is related to phospholipid peroxidation in mitochondria supplemented with Cu(II) and Fe(III).•Different response to metal addition indicates different intracellular toxicity mechanisms.•The direct impact on the organelle functionality of far lower concentrations of Cu than Fe suggests a dissociating event in the mechanism of toxicity of both metals.
Rat liver mitochondria (1.5–2.1mg protein·mL−1) supplemented with either 25 and 100μM Cu2+ or 100 and 500μM Fe3+ show inhibition of active respiration (O2 consumption in state 3) and increased ...phospholipid peroxidation . Liver mitochondria were supplemented with the antioxidants reduced glutathione, N-acetylcysteine or butylated hydroxitoluene, to evaluate their effects on the above-mentioned alterations. Although the mitochondrial dysfunction is clearly associated to phospholipid peroxidation, the different responses to antioxidant supplementation indicate that the metal ions have differences in their mechanisms of toxicity. Mitochondrial phospholipid peroxidation through the formation of hydroxyl radical by a Fenton/Haber-Weiss mechanism seems to precede the respiratory inhibition and to be the main fact in Fe-induced mitochondrial dysfunction. In the case of Cu2+, it seems that the ion oxidizes glutathione, and low molecular weight protein thiol groups in a direct reaction, as part of its intracellular redox cycling. The processes involving phospholipid peroxidation, protein oxidation and mitochondrial respiratory inhibition characterize a redox dyshomeostatic situation that ultimately leads to cell death. However, Cu2+ exposure involves an additional, yet unidentified, toxic event as previous reduction of the metal with N-acetylcysteine has only a minor effect in preventing the mitochondrial damage.
Cu2+ and Fe3+ overloads produce respiratory inhibition and phospholipid peroxidation in mitochondria. While the HO• formation by a Fenton/Haber-Weiss reaction is the responsible mechanism for Fe3+ cytotoxicity, Cu2+ also reacts with thiol groups and glutathione (GSH) since supplementation with GSH leads to full protection probably through formation of Cu(I)-(GSH)2 complexes. Display omitted
•Cu and Fe yield phospholipid oxidation and unpaired respiration in isolated mitochondria.•Fe and Cu showed differences in the mechanism of intracellular toxicity.•Lipid oxidation by hydroxyl radical is the main factor in Fe toxicity to mitochondria.•Cu may be oxidizing thiol groups in a direct reaction as part of its redox cycling.
The transition metals iron (Fe) and copper (Cu) are needed at low levels for normal health and at higher levels they become toxic for humans and animals. The acute liver toxicity of Fe and Cu was ...studied in Sprague Dawley male rats (200g) that received ip 0–60mg/kg FeCl2 or 0–30mg/kg CuSO4. Dose and time-responses were determined for spontaneous in situ liver chemiluminescence, phospholipid lipoperoxidation, protein oxidation and lipid soluble antioxidants. The doses linearly defined the tissue content of both metals. Liver chemiluminescence increased 4 times and 2 times after Fe and Cu overloads, with half maximal responses at contents (C50%) of 110μgFe/g and 42μgCu/g liver, and with half maximal time responses (t1/2) of 4h for both metals. Phospholipid peroxidation increased 4 and 1.8 times with C50% of 118μg Fe/g and 45μg Cu/g and with t1/2 of 7h and 8h. Protein oxidation increased 1.6 times for Fe with C50% at 113μg Fe/g and 1.2 times for Cu with 50μg Cu/g and t1/2 of 4h and 5h respectively. The accumulation of Fe and Cu in liver enhanced the rate of free radical reactions and produced oxidative damage. A similar free radical‐mediated process, through the formation HO• and RO• by a Fenton-like homolytic scission of H2O2 and ROOH, seems to operate as the chemical mechanism for the liver toxicity of both metals.
The acute liver toxicity of Fe and Cu overloads, evaluated from dose and time-responses, indicate that a similar chemical mechanism of a free radical‐mediated process seems to operate for the biochemical effects through the formation HO• and RO• by a Fenton-like homolytic scission of H2O2 and ROOH. Display omitted
► Liver oxidative damage is produced by higher than normal Fe and Cu liver contents. ► Metal concentration in liver is linearly related to metal intakes. ► Increased liver Fe and Cu contents similarly produced increased oxidative damage. ► This damage is simultaneous and generated for a common biochemical mechanism. ► Free radical process operate by HO• and RO• generated from H2O2 and ROOH scission.
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