Rescue of Cul3 haploinsufficiency phenotypes with CRISPR-mediated Cul3 activation

摘要 罕见和从头开始的单核苷酸变异(SNV)和拷贝数变异(CNV)是 神经发育障碍(NDDS)。大多数与NDD相关的SNV影响一个基因的单个等位基因， 导致单倍体不足。通过提高缺陷者的表达水平来纠正单倍性不足 等位基因为NDDS的治疗提供了一种有吸引力的策略。CRISPR的变种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 促进剂或增强剂。在这里，我们建议应用CRISPRA来上调CUL3泛素的水平 连接酶，NDDS的高置信度基因。我们最近建立了一个单倍体缺陷的CUL3小鼠模型。 脑部MRI发现皮质区域体积从出生后早期开始下降并持续 进入成年期。时空转录和蛋白质组学分析涉及细胞骨架和突触 缺陷是CUL3功能影响的关键驱动因素。具体地说，树突生长、丝状点状肌动蛋白和 多电极阵列(MEA)测得的自发网络活动在CUL3突变小鼠中降低。CUL3 突变的小鼠也表现出过度活跃的行为，以及社会和认知缺陷。我们假设 用CRSIPRa在发育早期上调CUL3剂量将挽救部分(或全部)观察到的 表型，并将奠定基础的一般治疗干预的NDDS。这个项目的目标是 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实现这一目标：(一) 评估CUL3+/-CRISPRA小鼠细胞和分子表型的挽救潜力；(2)评估 CUL3+/-CRISPRA小鼠脑结构和认知缺陷的挽救潜力。我们的研究将代表一种 重新平衡CUL3(和潜在的其他NDD基因)缺陷并纠正相关关联的有效策略 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.