Rescue of Cul3 haploinsufficiency phenotypes with CRISPR-mediated Cul3 activation

总结 罕见和从头单核苷酸变异（SNV）和拷贝数变异（CNV）是主要的风险因素， 神经发育障碍（NDD）。大多数NDD相关SNV影响基因的单个等位基因， 导致单倍不足。通过增加缺陷基因的表达水平来纠正单倍不足 等位基因可能为NDD治疗提供有吸引力的策略。CRISPR的变体CRISPRa（CRISPR 激活），提供了通过直接靶向内源基因的表达来调节内源基因表达的可能性。 启动子或增强子。在这里，我们建议应用CRISPRa来上调Cul 3泛素的水平。 连接酶，NDD的高置信度基因。我们最近产生了一个单倍不足Cul 3小鼠模型。 脑MRI发现皮质区体积减少，从出生后早期发育开始， 长大成人时空转录组学和蛋白质组学分析涉及细胞骨架和突触 缺陷作为Cul 3功能影响的关键驱动因素。具体来说，树突状生长，丝状肌动蛋白斑点， 在Cul 3突变小鼠中，通过多电极阵列（MEA）测量的自发网络活动减少。Cul3 突变小鼠也表现出多动行为，沿着社交和认知缺陷。我们假设 在发育早期用CRSIPRa上调Cul 3剂量将挽救一些（或全部）观察到的免疫缺陷。 表型，并将为NDD的一般治疗干预奠定基础。这个项目的目标 是为了证明通过重新平衡神经元，分子， 细胞和网络活动表型。我们将通过以下具体目标来实现这一目标：（1） 评估Cul 3 +/- CRISPRa小鼠中细胞和分子表型的拯救潜力;（2）评估 Cul 3 +/- CRISPRa小鼠的脑结构和认知缺陷的拯救潜力。我们的研究将代表一个 重新平衡Cul 3（以及潜在的其他NDD基因）缺陷和纠正相关缺陷的有效策略 表型

SUMMARY Rare and de novo single nucleotide variants (SNVs) and copy number variants (CNVs) are major risk factors for Neurodevelopmental Disorders (NDDs). The majority of NDD-associated SNVs affect a single allele of a gene, leading to haploinsufficiency. Correcting haploinsufficiency by increasing the expression level of the deficient allele could provide an attractive strategy for NDDs treatment. A variant of CRISPR, CRISPRa (CRISPR activation), offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters or enhancers. Here, we are proposing to apply CRISPRa to upregulate the levels of Cul3 ubiquitin ligase, a high-confidence gene for NDDs. We have recently generated a haploinsufficient Cul3 mouse model. Brain MRI found decreased volume of cortical regions starting from early postnatal development and persisting into adulthood. Spatiotemporal transcriptomic and proteomic profiling implicated cytoskeletal and synaptic defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity measured by multielectrode arrays (MEA) were reduced in Cul3 mutant mice. Cul3 mutant mice also exhibited hyperactive behavior, along with social and cognitive deficits. We hypothesize that upregulation of Cul3 dosage early in development with CRSIPRa will rescue some (or all) of the observed phenotypes, and will lay the basis for general therapeutic interventions in NDDs. The goal of this project is to demonstrate the feasibility of compensating for Cul3 haploinsufficiency by rebalancing neuronal, molecular, cellular and network activity phenotypes. We will achieve this goal through the following Specific Aims: (1) To evaluate rescue potential of cellular and molecular phenotypes in the Cul3+/- CRISPRa mice; (2) To evaluate rescue potential of brain architecture and cognitive deficits in Cul3+/- CRISPRa mice. Our study will represent an effective strategy for rebalancing Cul3 (and potentially other NDD genes) deficiency and correcting associated phenotypes.