Breasi-CRISPR Ccnd2 Thr280Ala mutation E13.5-E15.5 embryonic mouse cortex
Here we demonstrate the ability to model patient variants associated with two different NDDs in vivo using Breasi-CRISPR. We first model periventricular nodular heterotopia (PVNH), a neuronal migration disorder (Heinzen et al., 2018; Walters et al., 2018). We use Breasi-CRISPR to insert two patient analogous protein truncating variants in MAP1B, resulting in a significant defect in migration. We were able to validate that MAP1B fragments are being produced from these protein truncating variants, but the fragments do not act as a dominant negative. Next, we modeled a patient variant in CCND2 (encoding the protein cyclin D2) associated with megalencephaly postaxial polydactyly polymicrogyria hydrocephalus (MPPH) syndrome (Mirzaa et al., 2014). The unifying pathogenic mechanism underlying MPPH syndrome is believed to be excessive cyclin D2, a cell cycle regulator known to promote cell cycle progression. Modeling the repeated variant Thr280Ala caused an increase in the number of cells in S phase, an increase in progenitor numbers, an accumulation of cyclin D2 protein, and a tangential expansion of the cortex. We wondered if Breasi-CRISPR would be efficient enough to notice transcriptomic changes via bulk RNA sequencing of the targeted area. Indeed, we found that introduction of the MPPH syndrome variant caused an upregulation of genes and pathways important in tissue growth and a downregulation of those important in cell differentiation. Taken together we demonstrate that Breasi-CRISPR is efficient enough to model patient analogous variants, mirroring patient phenotypes and enabling investigation of NDDs. Overall design: Breasi CRISPR in utero electroporation inserting the Ccnd2 Thr280Ala variant in embryonic mouse brains was performed at E13.5, and RNA from the transfected regions was collected at E15.5