Department of Genetics, University Medical Center Utrecht Brain Center, University Medical Centre Utrecht, Utrecht, The Netherlands; and University Medical Centre Utrecht, Utrecht, The Netherlands; member of the ERN EpiCARE.
Department of Neurology and Neurosurgery, University Medical Centre Utrecht, Utrecht, The Netherlands; member of the ERN EpiCARE.
CRISPR J. 2021 Aug;4(4):575-582. doi: 10.1089/crispr.2021.0013.
Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene . DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published and loss-of-function models, we conclude that gain of function can have an equally severe phenotype. Upregulation of in the current zebrafish model for DS, -KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.
德拉维特综合征（DS）是一种单基因癫痫性脑病，由电压门控钠离子通道（VGSC）基因的功能丧失性突变引起。DS 的发病年龄在生命的第一年，且预后严重。在过去的几年中，已经表明在 DS 的动物模型中，内源性的上调可能是有益的，但没有观察到完全的挽救。我们假设在首发症状出现之前的早期发育过程中的上调可能会改善疾病的结局。为了验证这一假设，我们首先评估了 CRISPR 激活方法在早期发育过程中早期上调电压门控钠离子通道的作用。我们将靶向 VGSC 基因近端或远端启动子区域的 CRISPRa 组件注入单细胞期斑马鱼胚胎的卵黄中。评估了 dCas9-VPR 和 dCas9-VP64 的效果。没有或有向导 RNA 的情况下，这两种 CRISPRa 融合物在大多数胚胎中都表现出毒性。少数存活下来的胚胎发育正常，dCas9-VPR 在受精后 24 小时内诱导 mRNA 的上调。受精后 5 天，CRISPRa 注射的胚胎表现出癫痫表型，包括运动爆发运动、过度活跃和源自大脑的癫痫样活动。除了以前发表的 和 功能丧失模型外，我们得出结论，获得功能同样可以产生严重的表型。在当前的 DS 斑马鱼模型中上调 ， -KO，加重了疾病表型，这突出表明使用 CRISPRa 的早期上调既会导致毒性，也会导致疾病表型恶化。
